ISSR Marker Based Population Genetic Study of Melocanna … · 2019. 7. 30. · 2 Scientifica Nagaland Myanmar Mizoram Churachandpur Tamenglong Senapati Ukhrul N Chandel Imphal East

Research ArticleISSR Marker Based Population Genetic Study ofMelocanna baccifera (Roxb) Kurz A Commercially ImportantBamboo of Manipur North-East India

Heikrujam Nilkanta Thoungamba Amom Leimapokpam TikendraHamidur Rahaman and Potshangbam Nongdam

Department of Biotechnology Manipur University Canchipur Imphal Manipur 795003 India

Correspondence should be addressed to Potshangbam Nongdam purenbarediffmailcom

Received 6 August 2016 Accepted 4 December 2016 Published 10 January 2017

Academic Editor Massimo Ciccozzi

Copyright copy 2017 Heikrujam Nilkanta 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

Melocanna baccifera (Roxb) Kurz is an economically important bamboo of North-East India experiencing population depletionin its natural habitats Genetic variation studies were conducted in 7 populations sampled from 5 districts of Manipur using ISSRmolecular markers The investigation was carried out as a primary step towards developing effective conservation strategies forthe protection of bamboo germplasm ISSR marker analysis showed significant level of genetic variation within the populations asrevealed by moderately high average values of Neirsquos genetic diversity (H 01639) Shannonrsquos diversity index (I 02563) percentage ofpolymorphic bands (PPB 5918) total genetic variation (119867t 01961) and genetic diversity within population (119867s 01639) The studyalso divulged a high genetic variation at species level with Shannonrsquos diversity index (I) Neirsquos genetic diversity (H) and percentageof polymorphic band (PPB) recorded at 03218 01939 and 8837 respectively Genetic differentiation among the populations(119866st) was merely 1942 leaving 8058 of genetic variation exhibited within the populations The low genetic diversity betweenpopulations was consistent withAMOVAThe low genetic differentiation among populations coupledwith existence of significantlyhigh genetic diversity at species level indicated the urgent necessity of preserving and protecting all the existing natural bamboopopulations in the region

1 Introduction

Bamboos are multipurpose forest tree grasses having morethan 1000 different uses [1] They are extensively used as rawmaterials in paper and handicraft industries in house con-struction furniture making water pipes storage vessels andother important household items [2 3]Melocanna baccifera(Roxb) Kurz an evergreen arborescent non-clump formingbamboo is a dominant species of Manipur with its multipleuses in making of houses furniture handicraft and otherhousehold items [4 5]This bamboo also provides importantsupport for local ecosystem and is a priority species involvedin protection of local soils and biodiversity of forestedregions [6] Overexploitation of bamboo by locals in itsnatural reserves may lead to dramatic fall of populationThiswill result in great environmental degradation due to water

loss soil erosion and decline in natural biodiversity There-fore systematic management of depleting bamboo resourcethrough the adoption of proper conservation strategies witheither in situ or ex situ measures is the need of hour in theregion Assessing the level of genetic variation within andamong natural bamboo populations is highly crucial for thedevelopment of effective conservation methods [7ndash10] Thisis because the ability of a particular plant species to adapteffectively to changing environmental conditions dependson the level of genetic variability it possesses [11 12] DNAmolecular markers like random amplified polymorphic DNA(RAPD) inter-simple sequence repeats (ISSR) amplifiedfragment length polymorphism (AFLP) and simple sequencerepeat (SSR) have been used as influential molecular toolsfor determining genetic variation at species or populationlevel in different plants [13ndash18] The traditional approaches

HindawiScientificaVolume 2017 Article ID 3757238 9 pageshttpsdoiorg10115520173757238

2 Scientifica

Nagaland

Myanmar

Mizoram

Churachandpur

Tamenglong

Senapati

Ukhrul

N

Chandel

Imphal EastImphal West

ThoubalBishnupur

KilometersSampled area

0 100 200 300 400

Figure 1 Distribution and location of 7 populations ofM baccifera sampled in 5 districts (blackened area) of Manipur

of employing morphological and vegetative characters forbamboo species identification and genetic variation studieshad many shortcomings [19ndash21] However the applicationof RAPD ISSR AFLP SSR expressed sequence tag derived-simple sequence repeat (EST-SSR) sequence related ampli-fied polymorphism (SRAP) and restriction fragment lengthpolymorphism (RFLP) has enabled successful investigationof genetic variability in different bamboos [21ndash29] ISSRmolecular markers are widely used for population geneticanalysis of different plants generating more reliable andreproducible bands than RAPD [30 31] They are technicallysimpler as compared to RFLP SSR and AFLP markers asno previous sequence information is required for generat-ing DNA amplification products [32ndash34] There have beenlimited studies on the genetic variation of M bacciferausing ISSR markers and no reports are available on thepopulation genetic studies of the bamboo in Manipur Thepresent study aimed to investigate the genetic diversity andpopulation genetic structure of M baccifera in 5 districts ofManipur using ISSR markers This study could contributeto the development of strategies for effective conservationand sustainable utilization of this bamboo for ecological andeconomic gains by better understanding the genetic diversityprofile at the species and population level

2 Materials and Methods

21 Plant Materials and Population Sampling A total of 93individuals of M baccifera representing 7 populations were

collected from different locations spreading across 5 districtsofManipur inNorth-East India namely BishnupurThoubalImphal West Imphal East and Chandel (Figure 1) Thegeographical location of each population and its code nameand size were presented in Table 1 Fresh leaves were obtainedfrom bamboo plants constituting a particular populationThe sample collection was performed from individual plantsseparated by at least 50m so as to prevent any possibility ofsampling within the same clones The leaf samples were thenstored at minus20∘C until the DNA extraction was performed

22 DNA Extraction and ISSR Amplification The genomicDNA was extracted from the collected leaf samples usingthe CTAB method with slight modifications [35] The leavesafter being finely ground to fine powder in liquid nitrogenwere mixed with freshly prepared CTAB extraction bufferand incubated at 50∘C for 15ndash20 minutes in hot waterbath before being subjected to centrifugation at 12000 rpmfor 5 minutes The resultant supernatant was treated withchloroform isoamyl alcohol (24 1) followed by another cen-trifugation at 13000 rpm for 1-2 minutes The pellet obtainedafter 75M ammonium acetate treatment was washed severaltimes with 70 ice-cold ethanol and dried before beingresuspended in sterile DNase-free double distilled waterThe DNA sample obtained was further incubated at 65∘Cfor 20 minutes to destroy any DNase if present and storedat 4∘C for subsequent analysis DNA quality and quantitywere determined through spectrophotometry at 260 and280 nm respectively The purity and integrity were later

Scientifica 3

Table 1 Location of sample collection and sample size for seven populations ofM baccifera in the present study

Populationcode Locality Sample

size Latitude (N) Longitude (E) Elevation (m)

BISH Bishnupur district 14 24∘37101584087810158401015840 93∘451015840420510158401015840 828THBL-A Areas covering right to Indo-Myanmar road Thoubal district 14 24∘34101584079310158401015840 94∘2101584008610158401015840 790IMW-A Canchipur Sekmai Imphal West district 14 24∘361015840130310158401015840 94∘4101584007110158401015840 800IMPE Imphal East district 14 24∘45101584024810158401015840 93∘56101584007210158401015840 975CHDL Chandel district 14 24∘50101584098110158401015840 93∘541015840512210158401015840 1100IMW-B Kangchup areas Imphal West district 9 24∘471015840323810158401015840 94∘21015840186310158401015840 790

THBL-B Areas spanning forested regions to the left of Indo-Myanmar roadThoubal district 14 24∘271015840106410158401015840 94∘11015840330410158401015840 1500

checked by performing 10 agarose gel electrophoresis andcomparing the intensity of the resultant bands with 1 kb DNAladder (Hi-Media) The DNA samples were finally diluted to50 ng120583L and stored at minus20∘C for further use The geneticprofile study of M baccifera was conducted by using 5 ISSRmarkers namely UBC-813 UBC-822 UBC-828 UBC-868and UBC-878 obtained commercially from the University ofBritish Columbia (Vancouver Canada)The selected primersshowed good reliable repetitive and distinct bands whichenabled effective scoring for genetic diversity study withinand among the populations

The DNA amplification mixture of 25 120583L contained 25 ngtemplate DNA 1x PCR buffer 15mM MgCl2 200mMdNTPs 1 120583M primers (UBC-813 UBC-822 UBC-828 UBC-868 and UBC-878) and 06U Tag DNA polymerase anddouble distilled sterile water The PCR components wereprepared as master mix for each primer to minimize thepipetting error The amplification reaction was performed ina thermal cycler (Eppendorf Mastercycler nexus X2) withamplification cycle condition of initial 4 minutesrsquo strandsseparation at 94∘C followed by 40 cycles of 94∘C for 45 secs53∘C for 1min and 72∘C for 2 minutes and final extensionat 72∘C for 10 minutes The products obtained after PCRamplification were electrophoresed in 2 agarose gel in 05xTBE buffer at 100V for around 3 hours and gel was stainedwith ethidium bromide (05120583gmL) The fragments afterstaining were visualized in gel documentation system (AlphaInnotech USA) A 1000 bp DNA ladder (Hi-media) was usedas a size marker for every gel run

23 Data Analysis Distinct reproducible well resolved frag-ments were scored as present (1) or absent (0) for eachISSR reaction and were displayed as part of a binary matrixThe data matrices obtained were analyzed using POPGENEversion 131 [36] Genetic parameters such as percentage ofpolymorphic band(s) (PPB) observed number of alleles (119873a)the effective number of alleles (119873e) Neirsquos genetic diversity(119867) Shannonrsquos information index (119868) total genetic diversity(119867t) genetic diversity within population (119867s) Neirsquos geneticdifferentiation index among populations (119866st) and gene flowestimates between populations (119873m) were determined withPOPGENE version 131 [36] Genetic divergence betweenpopulations of the bamboo species was also investigatedusing Niersquos unbiased genetic distances and genetic identities

[37] A study on the correlation (Mantel test) between geneticand geographic distance between the bamboo populationswas done using TEPGA version 13 with 999 permutations[38] An analysis of molecular variance (AMOVA) wasperformed to estimate the variance components and their sig-nificance levels of genetic variation within and among popu-lations usingGenALEx version 65 [39]The unbiased geneticdistance was utilized for the construction of a dendrogramusing UPGMA (Unweighted Pair Group Arithmetic MeanMethod) in POPGENE program version 131 In additionNeirsquos genetic distancematrix was used to construct Neighbor-Joining dendrogram for the 93 individuals belonging to 7different populations of M baccifera using Mega 510 [40]Principal coordinate analysis (PCoA) was performed usingGenALEx version 65 [39] to determine relative geneticdistance between individuals and to check the consistencyof population genetic differentiation as defined by clusteranalysis The Bayesian model base clustering method ofSTRUCTURE software version 22 was employed to evaluatethe genetic population structure and detect the gene poolscontributing to bamboo germplasm collection [41] Ancestrymodel with admixture and correlated allele frequency modelwere used to determine the posterior probability of thedata and a burn in period of 10000 was set followed by10000 Markov Chain Monte Carlo (MCMC) replicationsThe number of 119870 was set from 1 to 8 with 10 independentruns for every119870 valueThe results of the STRUCTUREoutfilewere utilized to determine the optimum119870 value following thesimulation method of Evanno et al [42] employing softwareSTRUCTURE HARVESTER [43]

3 Results

31 Genetic Diversity The five different ISSR markers ampli-fied a total of 93 individuals representing 7 different popu-lations of M baccifera and generating high level of geneticpolymorphism High genetic variation at species level wasobserved in the present investigation with the recording ofpercentage of polymorphic bands (PPB) at 8837The valuesfor 119873a 119873e 119867 and 119868 at species level were also recordedat 18837 12751 01939 and 03218 respectively showing arelatively high level of genetic diversity (Table 2) Howeverthe genetic differentiation at population level was relativelylow as compared to genetic variation evidenced at species

4 Scientifica

Table 2 Genetic diversity within populations and genetic differentiation parameters of seven populations ofM baccifera

Population 119873a 119873e 119867 119868 PPB () 119867t 119867s 119866st 119873m

BISH 15581 11995 01320 02128 5581THBL A 16977 12411 01547 02504 6977IMW-A 15349 11626 01149 01927 5349IMPE 16279 12618 01678 02651 6279CHDL 16512 13923 02282 03400 6512IMW-B 15116 12914 01720 02594 5116THBL-B 15814 12888 01780 02737 5814Average 15940 12625 01639 02563 5918Species level 18837 12751 01939 03218 8837 Total 01961 01639 01942 25455119873a observed number of alleles119873e effective number of alleles119867 Neirsquos gene diversity 119868 Shannonrsquos information indices PPB percentage of polymorphic bands119867t total genetic diversity119867s genetic diversity within populations 119866st the relative magnitude of genetic differentiation among populations119873m estimate ofgene flow among populations

Table 3 Analysis of molecular variance (AMOVA) for five ISSR markers amongM baccifera populations

Source of variations Degree of freedom Sum of squares Mean square Variance components of total variance 119875 valueAmong populations 6 13687 2281 135 2200 lt0001Within populations 86 41992 488 488 7800 lt0001Total 92 55679 623 100

level This was proved by moderate PPB () recorded in therange of 5116 to 6977 averaging at 5918 Similarly theeffective number of alleles (119873e) varied from 11626 to 13923with an average value of 12625 while the observed numberof alleles (119873a) ranged from 15116 to 16977 averaging at 15940Neirsquos genetic diversity (119867) also extended from01149 to 02282with an average of 01639 and Shannonrsquos information index(119868) spanned from 01927 to 03400 with an average valueof 02563 THBL-A and CHDL exhibited highest geneticdiversity at population level while the lowest variation wasfound in IMW-B

32 Genetic Differentiation and Relationship among Popula-tions The genetic diversity within population (119867s) and thetotal genetic diversity (119867t) of the species were recorded at01639 and 01961 respectively The observed genetic differ-entiation among populations (119866st) was 01942 demonstratingthe presence of 1942 of genetic variation among thepopulations The finding was consistent with the result ofanalysis ofmolecular variance (AMOVA)which revealed 22of the genetic variation existing among populations and 78within the populations (Figure 2) The differences exhibitedbetween the populations were found to be highly significant(119875 = 0001) (Table 3) The result was further substantiated bythe existence of significantly high level of gene flowamong thepopulations as shown by high estimate of119873m value (25455)

Mantel test was performed to find out if there was anycorrelation between genetic relatedness between populationsand geographic distance separating them The test resultindicated that there was no significant correlation betweenthe genetic and geographic distance (119903 = 0311 119875 =0240) (Figure 3) The genetic distance obtained betweenthe 7 populations of M baccifera varied from 0011 betweenBISH and IMW-A to 0076 between CHDL and THBL-B

Among pops22

Within pops78

Figure 2 Analysis of molecular variance (AMOVA) revealed 22of the genetic variation existing among populations and 78 withinthe populations

20000 40000 60000 80000 1000000000Geographic distance (Km)

000000100020003000400050006000700080

Gen

etic

dist

ance

Figure 3 Correlation between genetic and geographic distanceamong 7 populations ofM baccifera

Scientifica 5

Table 4 Neirsquos unbiased measures of genetic identity (above diagonal) and genetic distance (below diagonal) of the seven populations ofMbaccifera

BISH THBL-A IMW-A IMPE CHDL IMW-B THBL-B Populationlowast lowast lowast lowast lowastlowast 0986 0989 0985 0958 0948 0984 BISH0014 lowast lowast lowast lowast lowast lowast lowast 0983 0982 0967 0949 0962 THBL-A0011 0017 lowast lowast lowast lowast lowast lowast lowast 0985 0942 0945 0977 IMW-A0015 0018 0015 lowast lowast lowast lowast lowast lowast lowast 0952 0940 0980 IMPE0043 0033 0060 0049 lowast lowast lowast lowast lowast lowast lowast 0936 0927 CHDL0054 0052 0056 0062 0066 lowast lowast lowast lowast lowast lowast lowast 0939 IMW-B0016 0039 0023 0021 0076 0063 lowast lowast lowast lowast lowast lowast lowast THBL-B

BISHIMW-ATHBL-AIMPETHBL-BCHDLIMW-B

000000050010001500200025

Figure 4 UPGMA dendrogram based on Neirsquos (1972) unbiasedmeasures of genetic distance among 7 populations ofM baccifera

respectively (Table 4)The dendrogramdisplay based onNeirsquos[44] unbiased measures of genetic distances showed onlyone major cluster comprising of BISH IMW-A THBL-AIMPE CHDL and THBL-B The CHDL population tendedto separate from the cluster indicating its genetic isolationfrom other populations in the group (Figure 4) But CHDLexhibited more genetic proximity to THBL-B as comparedto other remaining populations in the same cluster withthe recording of significantly high value of Neirsquos unbiasedmeasures of genetic identity (0967) The existing greatergenetic identity between CHDL and THBL-B in spite ofindividuals of CHDL being sampled from hilly dominatedregion may be attributed to geographic closeness of thetwo populations The lone IMW-B population diverged fromthe major cluster in the dendrogram depicting its lessgenetic relatedness to remaining populations existing in themajor cluster Neighbor-Joining radial tree obtained for theindividual samples based on Neirsquos genetic distance showedmost of the plants belonging to different population originsseparated distinctly though some individuals were partlymixed clustered (Figure 5) Individual genotypes belongingto same populationweremore closely related genetically thanthose of other populations

The principal coordinate analysis (PCoA) was conductedto determine the spatial representation of genetic distancesobserved among individuals of different populations and alsoto check the consistency of population genetic differentiationas defined by cluster analysis The two-dimensional PCoAplot showed the first principal coordinate accounting for1632 while the second coordinate produced 1090 of thetotal genetic variation The individuals of seven bamboo

populations were distributed in the plot in accordance withthe cluster analysis The individuals belonging to CHDL andIMW-B were scattered separately from the rest of the popu-lations (Figure 6) The remaining populations were clusteredas one group with individuals more or less intermixed as wasdepicted in the cluster analysis

The highest delta 119870 which determined the best valueof 119870 was obtained from STRUCTURE HARVESTER andwas found to be shown at 119870 = 3 (Figure 7) The resultindicated that all the 93 individuals of seven populations ofM baccifera shared three genetic pools and were assignedinto three different clusters (Figure 8)The seven populationsdisplayed significant degree of mixed ancestry as representedin the structure diagram

4 Discussion

The genetic variation analysis conducted in 7 populationsof M baccifera revealed the existence of high genetic diver-sity within population which was similar to earlier find-ing in Dendrocalamus membranaceus using ISSR markers[28] Plants which are long lived outcrossing and self-incompatible have higher genetic variation at species leveland lower differentiation among populations [11 45] Thehigh genetic variation within the population of M bacciferawas expected as being a long lived woody bamboo withlong vegetative phase [46] Self-incompatibility is anotherimportant factor in maintaining high genetic variabilityin population [47] and M baccifera is known to be self-incompatible and outcrossing bamboo thereby displayingsignificant genetic diversity within population Nybom [48]also reported retention of most of genetic variability withinpopulations of long lived and outcrossing plant specieswhen many RAPD and sequence tagged microsatellite site(STMs) based analysis were performed The plants with highgeographical ranges tend tomaintain higher genetic diversitythan geographically localized species [49] M baccifera isknown to show diverse distribution pattern along the variousgeographical locations of Manipur [6] Genetic diversitywithin populations is also influenced by many factors suchas mating system population size extended time periodwith low number of individuals genetic drift and gene flow[50] High genetic diversity within small populations canalso be exhibited if reduction of population size had takenplace very recently especially when it occurred within ageneration or two for the concerned species [51] In such

6 Scientifica

IMW

-A7

IMPE

4BI

SH1

0IM

PE3

IMPE

1IM

PE2

IMW

-A14

THBL

-A10

BISH

11IM

W-A

8CHDL1

CHDL2

BISH1

BISH13

IMPE5

THBL-B14

IMW-A6

IMW-A1

IMW-A3

IMW-A2

IMW-A5

BISH9

THBL-B8

THBL-B10THBL-B11BISH8IMW-A11BISH6

IMW-B8THBL-A9THBL-A11THBL-A12THBL-A13

THBL-A6IMW-A10

THBL-A7THBL-A5

IMW-A9

IMW-A12

IMW

-A13

THBL-A14

IMPE14

BISH3

BISH7

BISH2

BISH12

BISH14IM

PE8

IMPE

10

IMPE

6IM

PE9

IMPE

7

BISH

4

IMW

-B9

THBL

-B12

THBL

-B13

THBL

-B9

THBL

-B1

THBL-B2

THBL-B5

THBL-B7

THBL-B6THBL-B3

THBL-B4IMPE13IMPE11IMPE12BISH5CHDL6THBL-A1THBL-A8THBL-A4THBL-A3IMW-B7

CHDL3IMW-B1

IMW-B2

IMW-B3

IMW-B6

IMW-B4

IMW-B5

CHDL11

CHDL4

CHDL9

CHDL5

THBL-A2

CHDL7CHDL14

CHDL10CHDL8

CHDL12CH

DL13IM

W-A4

Figure 5 Neighbor-Joining dendrogram clustering pattern for 93 individuals belonging to 7 populations ofM baccifera

Coo

rd 2

Coord 1

Principal coordinates (PCoA)

BISHTHBL-AIMW-AIMPE

CHDLIMW-BTHBL-B

Figure 6 Two-dimensional plot of principal coordinate analysis(PCoA) showing clustering of individual samples belonging to 7populations ofM baccifera

cases the surviving individuals are effectively sampled fromthe populations that have existed before The existence ofsignificant genetic variation within the populations of Mbaccifera under study may also be due to sudden reduction in

4035 45 50 55 6030

0

10

20

30

40

Delt

a K

Delta K = mean (IL998400998400(K)I)sd(L(K))

Figure 7 Result of Bayesian assignment analysis suggesting 119870 = 3as most likely number of clusters as delta 119870 value maximum at 119870 =3

population size in short span of timeThis presumptionmighthold true asmore accessible individuals growing in the regionhave been exploited by locals for construction of houses andother household products along with the utilization of theirtender shoots as popular vegetable items

Scientifica 7

BISH

THBL

-A

IMW

-A

IMPE

CHD

L

IMW

-B

THBL

-B

100080060040020000

Figure 8 Population structure pattern of 7 populations of Mbaccifera generated using STRUCTURE program when 119870 = 3

The coefficient of genetic differentiation among popu-lations (119866st) was 01942 which was higher than the meanvalue (119866st 0073) observed for 121 woody species exam-ined using allozyme markers [52] Yang et al [28] alsorecorded low 119866st (0252) in ISSR analysis of Dendrocalamusmembranaceus populations in Yunnan province of ChinaThe low 119866st value recorded for M baccifera indicated lowlevel of genetic differentiation among the populations Lowgenetic differentiation between populations had also beenconfirmed by AMOVA (analysis of molecular variance) Thefinding was also consistent with high 119873m (25455) recordedwhich was much higher than 119873m (00101) recorded forDendrocalamus giganteus by Tian et al [21] The geneticdifferentiation between populations is negatively correlatedwith gene flow [53]Gene flowbetween the populationsmighthave been induced by pollens and seeds highly influencingplant evolution [54] High gene flow (119873m 2545) was observedin spite of poor seed dispersal mechanism of M bacciferaThe flower cycle of this bamboo is relatively long and largeseeds which fall near the foot of plants are mostly eatenby rats and rodents However M baccifera being one ofthe economically important bamboos of the region mighthave undergone human mediated movement of genotypesthrough local people farmers and entrepreneurs This maylead to enhancement of gene flow among populations andproduce overlapping and intermixing of bamboo genotypesfromdifferent populationsThe easy geneticmovementmightalso be possible as the regions of population sampling in thepresent study were confined mostly to plain valley area Infact majority of the populations under study were locatedin valley districts except for the one population sampledfrom hill district of Chandel The STRUCTURE result alsorevealed high degree of admixture among the 7 populationsin three genetic clusters The existence of great extent ofadmixture may be attributed to cultivation and utilizationpattern of the bamboo species Geographic isolation isanother major factor influencing the genetic differentiationamong populations by limiting the amount of gene flow viaboth pollen and seeds and also through human activities[55] Theoretically gene flow of more than four migrantsper generation is sufficient to prevent genetic differentiationbetween populations due to drift alone In our study thegene flow estimate was high (119873m 25455) ruling out thepossibility of inducing the genetic differentiation amongpopulations of M baccifera due to geographical isolationsThe results from the Mantel test also supported the above

observation as it showed no correlation between the geneticand geographic distances between different populations Theultimate goal of conservation is to ensure the continuoussurvival of population and to maintain their evolutionarypotential by preserving natural levels of genetic diversitywithin and between populations [9 56 57] The natural Mbaccifera resources in the selected sites have been exploitedby locals for many years leading to loss of plant resourceshabitat destruction and fragmentation The dramatic fallof bamboo natural population due to increasing demandfor its multiutility characters ensures that there is urgentrequirement of effective conservation The population sizeof M baccifera sampled from different locations was smallrequiring population enhancement to maintain standardeffective size for high genetic diversity preservation Geneticdrift in very small population might also cause rapid geneticerosion and increased the risk of extinction of the bamboospecies Considering theM bacciferamaintaining significantdiversity at species level but low genetic variation amongpopulations and thatmany native habitats and ecosystems arebeing destroyed by human interferences great effort shouldbe made to preserve individuals of the existing populationsExploitation of bamboos and habitat destruction should beprohibited in the region till effective in situ and ex situconservation strategies are established This will preventfurther reduction of population size and conserve the overallgenetic base and structure of the bamboo species The insitu efforts to conserve the remaining habitats should becombined with ex situ approach through seed and vegetativepropagation of culm cutting division and tissue culturetechnologyThese activities should be performed with a viewto establishing a new generation of plants both cultivableand wild A more effective protection strategy may also beframed especially for BISH IMW-A IMW-B and THBL-B which displayed low genetic diversity at population levelSensitization of locals about the prevailing genetic scenarioof alarming bamboo populations and involvement of localcommunities in framing protection policies will be a highlyeffective approach to bamboo conservation The presenceof high genetic variation within populations of M bacciferaemphasizes the necessity of preserving and conserving all theexisting 7 natural populations and their habitats in Manipur

Competing Interests

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

Acknowledgments

The authors are thankful to SERB (Science Engineering andResearch Board) New Delhi India for providing financialsupport

References

[1] A Lewington Plant for People Natural History Museum Pub-lications London UK 1990

8 Scientifica

[2] M Das S Bhattacharya P Singh T S Filgueiras and A PalldquoBamboo taxonomy and diversity in the era of molecularmarkersrdquo Advances in Botanical Research vol 47 pp 225ndash2682008

[3] P Nongdam and L Tikendra ldquoThe nutritional facts of bambooshoots and their usage as important traditional foods of north-east Indiardquo International Scholarly Research Notices vol 2014Article ID 679073 17 pages 2014

[4] H B Naithani ldquoDiversity of bamboo species in North-eastIndiardquo in Ecology Diversity and Conservation of Plants andEcosystems in India H N Pandey and S K Barik Eds pp 312ndash322 Regency Publication New Delhi India 2006

[5] J M S Tomar D K Hore and A Annadurai ldquoBamboo andtheir conservation in North east Indiardquo Indian Forester vol 6pp 817ndash824 2009

[6] H BNaithaniN S Bisht and S SingsitDistribution of BambooSpecies in Manipur Principle Chief Conservation of ForestManipur India 1st edition 2010

[7] J Liu S Shi E ChangW Yang and Z Jiang ldquoGenetic diversityof the critically endangered Thuja sutchuenensis revealed byISSR markers and the implications for conservationrdquo Interna-tional Journal of Molecular Sciences vol 14 no 7 pp 14860ndash14871 2013

[8] Z QMei S Y Fu H Q Yu et al ldquoGenetic characterization andauthentication of Dimocarpus longan Lour using an improvedRAPD techniquerdquo Genetics and Molecular Research vol 13 no1 pp 1447ndash1455 2014

[9] P Desai B Gajera M Mankad et al ldquoComparative assessmentof genetic diversity among Indian bamboo genotypes usingRAPD and ISSR markersrdquo Molecular Biology Reports vol 42no 8 pp 1265ndash1273 2015

[10] L YeasminMN Ali S Gantait and S Chakraborty ldquoBambooan overview on its genetic diversity and characterizationrdquo 3Biotech vol 5 no 1 pp 1ndash11 2015

[11] J L Hamrigk and M J W Godt ldquoEffects of life history traitson genetic diversity in plant speciesrdquo Philosophical Transactionsof the Royal Society B Biological Sciences vol 351 no 1345 pp1291ndash1298 1996

[12] R Frankham ldquoGenetic adaptation to captivity in species con-servation programsrdquo Molecular Ecology vol 17 no 1 pp 325ndash333 2008

[13] S-M Chung J E Staub and J-F Chen ldquoMolecular phylogenyof Cucumis species as revealed by consensus chloroplast SSRmarker length and sequence variationrdquo Genome vol 49 no 3pp 219ndash229 2006

[14] D Ngrsquouni M Geleta and T Bryngelsson ldquoGenetic diversity insorghum (Sorghum bicolor (L) Moench) accessions of Zambiaas revealed by simple sequence repeats (SSR)rdquo Hereditas vol148 no 2 pp 52ndash62 2011

[15] G Della Rocca T Osmundson R Danti et al ldquoAFLP analy-ses of california and mediterranean populations of Seiridiumcardinale provide insights on its origin biology and spreadpathwaysrdquo Forest Pathology vol 43 no 3 pp 211ndash221 2013

[16] J Sabir M Mutwakel A El-Hanafy et al ldquoApplying moleculartools for improving livestock performance from DNAmarkersto next generation sequencing technologiesrdquo Journal of FoodAgriculture and Environment vol 12 no 2 pp 541ndash553 2014

[17] M Basahi ldquoISSR-based analysis of genetic diversity amongsorghum landraces growing in some parts of Saudi Arabia andYemenrdquo Comptes Rendus Biologies vol 11 no 338 pp 723ndash7272015

[18] J Gao W Zhang J Li H Long W He and X Li ldquoAmplifiedfragment length polymorphism analysis of the populationstructure and genetic diversity of Phoebe zhennan (Lauraceae)a native species to Chinardquo Biochemical Systematics and Ecologyvol 64 pp 149ndash155 2016

[19] D Ohrnberger and J Goerrings The Bamboos of the WorldElsevier Odenthal Germany 1986

[20] D Fan X Jiarong Y Yuming et al ldquoStudy on floweringphenomenon and its type of bamboo in Yunnan in past fifteenyearsrdquo Scientia Silvae Sinica vol 3 pp 57ndash68 2000

[21] B Tian H-Q Yang K-M Wong A-Z Liu and Z-YRuan ldquoISSR analysis shows low genetic diversity versus highgenetic differentiation for giant bambooDendrocalamus gigan-teus (Poaceae Bambusoideae) in China populationsrdquo GeneticResources and Crop Evolution vol 59 no 5 pp 901ndash908 2012

[22] E Friar and G Kochert ldquoA study of genetic variation and evo-lution of Phyllostachys (Bambusoideae Poaceae) using nuclearrestriction fragment length polymorphismsrdquo Theoretical andApplied Genetics vol 89 no 2-3 pp 265ndash270 1994

[23] C Lai and J Y Hsiao ldquoGenetic variation of Phyllostachyspubescens (Bambusoideae Poaceae) in Taiwan based on DNApolymorphismsrdquo Botanical Bulletin of Academia Sinica vol 38no 3 pp 145ndash152 1997

[24] T RHodkinson S A Renvoize GN Chonghaile CMA Sta-pleton and M W Chase ldquoA comparison of ITS nuclear rDNAsequence data and AFLP markers for phylogenetic studiesin Phyllostachys (Bambusoideae Poaceae)rdquo Journal of PlantResearch vol 113 no 3 pp 259ndash269 2000

[25] N A Barkley M L Newman M L Wang M W Hotchkissand G A Pederson ldquoAssessment of the genetic diversity andphylogenetic relationships of a temperate bamboo collection byusing transferred EST-SSRmarkersrdquoGenome vol 48 no 4 pp731ndash737 2005

[26] S Bhattacharya M Das R Bar and A Pal ldquoMorphologicalandmolecular characterization of bambusa tulda with a note onfloweringrdquo Annals of Botany vol 98 no 3 pp 529ndash535 2006

[27] R K Sharma P Gupta V Sharma A Sood T Mohapatraand P S Ahuja ldquoEvaluation of rice and sugarcane SSR markersfor phylogenetic and genetic diversity analyses in bamboordquoGenome vol 51 no 2 pp 91ndash103 2008

[28] H-Q Yang M-Y An Z-J Gu and B Tian ldquoGenetic diversityand differentiation of Dendrocalamus membranaceus (PoaceaeBambusoideae) a declining bamboo species in Yunnan Chinaas based on Inter-Simple Sequence Repeat (ISSR) analysisrdquoInternational Journal of Molecular Sciences vol 13 no 4 pp4446ndash4447 2012

[29] S Zhu T Liu Q Tang L Fu and S Tang ldquoEvaluation ofbamboo genetic diversity usingmorphological and SRAPanaly-sesrdquoRussian Journal of Genetics vol 50 no 3 pp 267ndash273 2014

[30] T Nagaoka and Y Ogihara ldquoApplicability of inter-simplesequence repeat polymorphisms inwheat for use asDNAmark-ers in comparison to RFLP and RAPDmarkersrdquoTheoretical andApplied Genetics vol 94 no 5 pp 597ndash602 1997

[31] L-J Zhang and S-L Dai ldquoGenetic variation within and amongpopulations ofOrychophragmus violaceus (Cruciferae) inChinaas detected by ISSR analysisrdquo Genetic Resources and CropEvolution vol 57 no 1 pp 55ndash64 2010

[32] S P Joshi V S Gupta R K Aggarwal P K Ranjekar andD S Brar ldquoGenetic diversity and phylogenetic relationship asrevealed by inter simple sequence repeat (ISSR) polymorphismin the genus Oryzardquo Theoretical and Applied Genetics vol 100no 8 pp 1311ndash1320 2000

Scientifica 9

[33] B Bornet and M Branchard ldquoNonanchored inter simplesequence repeat (ISSR)markers reproducible and specific toolsfor genome fingerprintingrdquo Plant Molecular Biology Reportervol 19 no 3 pp 209ndash215 2001

[34] M X Wang H L Zhang D L Zhang et al ldquoGenetic structureof Oryza rufipogon Griff in ChinardquoHeredity vol 101 no 6 pp527ndash535 2008

[35] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf materialrdquo Phytochemical Bulletinvol 19 pp 11ndash15 1987

[36] C F Yeh R Yang and T Boyle POPGENE VERSION 131Microsoft Windows-Based Freeware for Population GeneticsAnalysis University of Alberta and Centre for InternationalForestry Research Alberta Canada 1999

[37] M Nei ldquoEstimation of average heterozygosity and genetic dis-tance from a small number of individualsrdquo Genetics vol 89 no3 pp 583ndash590 1978

[38] M P Miller Tools for Populations Genetic Analyses (TFPGA)Version 13 A Windows Program for the Analysis of Allozymesand Molecular Population Genetic Data Utah State UniversityLongan Utah USA 1997

[39] R Peakall and P E Smouse ldquoGenAlEx 65 genetic analysis inExcel Population genetic software for teaching and researchmdashan updaterdquo Bioinformatics vol 28 no 19 pp 2537ndash2539 2012

[40] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[41] J K Pritchard M Stephens and P Donnelly ldquoInference ofpopulation structure using multilocus genotype datardquoGeneticsvol 155 no 2 pp 945ndash959 2000

[42] G Evanno S Regnaut and J Goudet ldquoDetecting the numberof clusters of individuals using the software STRUCTURE asimulation studyrdquo Molecular Ecology vol 14 no 8 pp 2611ndash2620 2005

[43] D A Earl and B M VonHoldt ldquoStructure Harvester a websiteand program for visualizing Structure output and implementingthe Evanno Methodrdquo Conservation Genetics Resources vol 4no 2 pp 359ndash361 2012

[44] M Nei ldquoGenetic distance between populationsrdquoThe AmericanNaturalist vol 106 no 949 pp 283ndash292 1972

[45] G Zawko S L Krauss K W Dixon and K SivasithamparamldquoConservation genetics of the rare and endangered Leucopogonobtectus (Ericaceae)rdquo Molecular Ecology vol 10 no 10 pp2389ndash2396 2001

[46] S Jeeva S Kiruba H Lahruaitlung et al ldquoFlowering ofMelocanna baccifera (Bambusaceae) in North-eastern IndiardquoCurrent Science vol 96 pp 1165ndash1166 2009

[47] E L Borba J Semir and G J Shepherd ldquoSelf-incompatibilityinbreeding depression and crossing potential in five BrazilianPleurothallis (Orchidaceae) speciesrdquo Annals of Botany vol 88no 1 pp 89ndash99 2001

[48] H Nybom ldquoComparison of different nuclear DNAmarkers forestimating intraspecific genetic diversity in plantsrdquo MolecularEcology vol 13 no 5 pp 1143ndash1155 2004

[49] L Chen F Chen S He and L Ma ldquoHigh genetic diversity andsmall genetic variation among populations ofMagnolia wufen-gensis (Magnoliaceae) revealed by ISSR and SRAP markersrdquoElectronic Journal of Biotechnology vol 17 no 6 pp 268ndash2742014

[50] S George J Sharma and V L Yadon ldquoGenetic diversity ofthe endangered and narrow endemic Piperia Yadonii (Orchi-daceae) assessed with ISSR polymorphismsrdquo American Journalof Botany vol 96 no 11 pp 2022ndash2030 2009

[51] X Zhao Y Ma W Sun X Wen and R Milne ldquoHigh geneticdiversity and low differentiation of Michelia coriacea (Magno-liaceae) a critically endangered endemic in southeast YunnanChinardquo International Journal of Molecular Sciences vol 13 no4 pp 4396ndash4411 2012

[52] J L Hamrick M J W Godt and S L Sherman-BroylesldquoFactors influencing levels of genetic diversity in woody plantspeciesrdquo New Forests vol 6 no 1 pp 95ndash124 1992

[53] V Grant The Evolutionary Process A Critical Study of Evolu-tionaryTheory ColumbiaUniversity PressNewYorkNYUSA1991

[54] J L Hamrick ldquoGene flow and distribution of genetic variationin plant populationsrdquo in Differentiation Patterns in HigherPlants K Urbanska Ed pp 53ndash76 Academic Press New YorkNY USA 1987

[55] M Pfeifer and G Jetschke ldquoInfluence of geographical isola-tion on genetic diversity of Himantoglossum hircinum (orchi-daceae)rdquo Folia Geobotanica vol 41 no 1 pp 3ndash20 2006

[56] Z-Y Zhang Y-Y Chen and D-Z Li ldquoDetection of low geneticvariation in a critically endangered Chinese pine Pinus squa-mata using RAPD and ISSR markersrdquo Biochemical Geneticsvol 43 no 5-6 pp 239ndash249 2005

[57] H Wu Z Li and H Huang ldquoGenetic differentiation amongnatural populations of Gastrodia elata (Orchidaceae) in Hubeiand germplasm assessment of the cultivated populationsrdquoBiodiversity Science vol 14 no 4 pp 315ndash326 2006

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

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International Journal of

Volume 2014

Zoology

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Molecular Biology International

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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