Journal of Genetic Engineering and Biotechnology (2012) 10, 39–45

Academy of Scientific Research & Technology andNational Research Center, Egypt

Journal of Genetic Engineering and Biotechnology

www.elsevier.com/locate/jgeb

ARTICLE

Assessment of genetic diversity in Lepidium sativum(Chandrasur) a medicinal herb used in folklore

remedies in India using RAPD

Dinesh Bansala, Pragya Bhasin

a, O.P. Yadav

b, Anita Punia

a,*

a Department of Biotechnology, University Institute of Engineering & Technology, Kurukshetra University, Kurukshetra 136119,Haryana, Indiab Medicinal and Aromatic Plants Section, C.C.S. Haryana Agricultural University, Hisar, Haryana, India

Received 29 March 2012; revised 9 April 2012; accepted 14 April 2012Available online 15 May 2012

*

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16

Pr

Pe

ht

KEYWORDS

Lepidium sativum

(Chandrasur);

RAPD;

PCR;

Genetic diversity;

PCA;

Polymorphism

Corresponding author.

mail address: anitapunia_17@

87-157X ª 2012 Academy

oduction and hosting by Els

er review under National Re

tp://dx.doi.org/10.1016/j.jgeb

Production and h

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Abstract Lepidium sativum L. is a fast growing edible herb which belongs to family Brassicaceae.

The seeds of L. sativum are aperient, diuretic, tonic, demulcent, carminative, galatogogue and

emmenagogue. They have been used in the treatment of bacterial and fungal infections, as an ape-

rient and also possess antibacterial and antifungal properties. The seeds of this plant possess rapid

bone fracture healing ability. Despite of its diverse medicinal properties no molecular data for diver-

sity analysis is available till date. During this study random amplified polymorphic DNA (RAPD)

markers were used to detect genetic variations of L. sativum. Initially 50 decamer primers were

screened, out of which only 32 primers showed reproducible fragments with easily recordable

bands. A total of 414 reproducible and clear bands were distinguished across the selected primers

and statistical analysis showed 361 polymorphic bands and 53 monomorphic bands. Cluster anal-

ysis of the genotypes based on UPGMA divided the 18 genotypes into two main clusters, with first

cluster having only HCS-20 genotype of L. sativum and other having rest of all 17 genotypes. The

dendrogram based on similarity matrix revealed 23–66% genetic relatedness among 18 genotypes.

ail.com (A. Punia).

ific Research & Technology.

. All rights reserved.

enter, Egypt.

002

lsevier

Table 1 DNA amplification bands

S.No. Primer code Primer

1 6800-016 TGGA

2 6800-017 CCCA

3 6800-018 GGAA

4 6800-019 CAGC

5 6800-021 GGGA

6 6800-023 AAGC

7 6800-024 GTCC

8 6800-025 GGTT

9 6800-026 TCGG

10 6800-028 TTCG

11 6800-031 CCAA

12 6800-032 AGGC

13 6800-033 CAAA

14 6800-034 GGAG

15 6800-035 GACA

16 6800-037 GACA

17 6800-038 CACC

18 6800-043 GTGA

19 6800-044 GTGG

20 6800-045 GGAA

21 6800-048 CAAG

22 6800-049 GTGG

23 6800-050 CAGA

24 6800-054 CCGA

25 6800-055 GTAA

26 6800-056 GGAG

27 6800-057 ACCT

28 6800-058 GTAG

29 6800-059 TCGT

30 6800-060 TGTA

31 6800-061 AC G

32 6800-063 GTGT

Bold letters indicate the maximum and

40 D. Bansal et al.

The results of the present study can be used for molecular breeding and improvement of L. sativum

for various desired traits through hybridization in future.

ª 2012 Academy of Scientific Research & Technology. Production and hosting by Elsevier B.V.

All rights reserved.

1. Introduction

Medicine in several developing countries, using local traditions

and beliefs, is still the mainstay of health care. Ayurveda re-mains an important system of medicine and drug therapy inIndia. Plant alkaloids are the primary active ingredients ofAyurvedic drugs [11]. The need for preservation of these valu-

able genetic resources creates an incentive for determination ofthe genetic variability present among these plant species usingmolecular and biochemical markers.

Lepidium sativum L. is a fast growing edible herb the genusconsists of approximately 175 species; some of which are culti-vated which include garden cress (L. sativum), Maca (Lepidium

meyenii), Walp (Lepidium peruvianum). Chandrasur is the oneingredient of compound substance called Chaturbija, whichindicates that especially the seeds were esteemed for theirmedicinal properties. The seeds of L. sativum are aperient,

diuretic, tonic, demulcent, carminative, galatogogue, emmena-gogue, are used to procure an abortion, and also possess anti-

and polymorphism generated in

sequence Genotypes

amplified

CTCGGT 9

AGCGAA 8

CGCTAC 11

GGGTCA 17

AGCGTC 14

GGCCCT 16

TGCTGT 17

GTTCCC 14

TGAGTC 17

GCGATG 17

GCCGTC 13

CAACAG 15

GGCGTG 10

CTGACT 12

CACTCC 17

GGTTGG 8

CCGAAA 11

CCAGAG 16

CTCTCC 17

AGCCCA 16

CCGTGA 17

ATCGTC 7

GGTTCC 7

CTCTGG 12

GCCGAG 16

CAGCAA 8

GCCAAC 10

GTCGCA 14

GGCACA 11

CGGCAC 15

GAGGCAG 13

GCCTGG 7

minimum values.

bacterial and antifungal properties [10]. Raval and Pandya [16]conducted clinical trials of L. sativum and showed that it pro-vides very good relief in cardinal signs, symptoms like pain in

joint, swelling, stiffness, crepitus, tenderness and difficulty inmovement. The gum produced from this plant has high molec-ular weight [8]. It has various characteristic like binding, disin-

tegrating, gelling etc. [7].Molecular markers have been used in determining genetic

diversity and to reconstruct evolutionary processes [23]. In

contrast to the traditional selection based on phenotypicscreening, molecular markers refractory to environmental var-iation are fully heritable and available in large numbers. In or-der to develop an efficient identification method, molecular

techniques have been used since these are reliable, unaffectedby environmental conditions and can aid varietal identifica-tion. Among the different types of molecular markers avail-

able, random amplified polymorphic DNA (RAPD) is usefulfor the assessment of genetic diversity among rare species [3].RAPD markers have many advantages like the requirement

Lepidium sativum genotypes using random primers.

Total

bands

Polymorphic Monomorphic % Age

polymorphic

9 8 1 88.89

3 2 1 66.67

10 10 0 100

12 9 3 75

6 5 1 83.33

16 15 1 93.75

18 15 3 83.33

25 25 0 100

8 7 1 87.5

15 14 1 93.33

9 9 0 100

11 9 2 81.82

10 9 1 90

11 10 1 90.91

5 4 1 80

11 9 2 81.82

16 12 4 75

10 8 2 80

17 16 1 94.12

16 13 3 81.25

15 13 2 86.67

19 18 1 94.74

10 8 2 80

9 7 2 77.78

18 12 6 66.67

15 13 2 86.67

11 9 2 81.82

17 17 0 100

24 23 1 95.83

13 13 0 100

5 4 1 80

20 15 5 75

Figure 1 RAPD pattern of different varieties of L. sativum

amplified with 6800-18 when subjected to 1.8% Agarose Gel

Electrophoresis stained with ethidium bromide. Lane M represents

molecular weight marker. Lane 1 (HCS1), 2 (HCS2), 3(HCS-3),4

(HCS-4), 5 (HCS-5), 6 (HCS-6), 7 (HCS-7), 8 (HCS-8), 9(HCS-9),

10(HCS-10), 11(HCS-11), 12 (HCS12), 13 (HCS-13), 14 (HCS-

14),15 (HCS-15), 16 (HCS-16), 17 (HCS-17), 18 (HCS-18),

respectively.

Assessment of genetic diversity in Lepidium sativum (Chandrasur) a medicinal herb 41

of very small quantity of template DNA and no involvementof radioactive or hybridization techniques. RAPD markershave been extensively used in constructing linkage maps,

assessment of genetic variation in population, gene taggingand identification of cultivars and species [19,21] despite ofthe existing limitation of dominant mode of inheritance of

RAPD loci. Todate, no universal marker system is availablefor codominant markers, which is applicable to every speciessuch markers need to be developed separately for each species

and can be extremely costly in resources and time [12]. Geneticpolymorphisms naturally prevailing in plants has been studiedwidely and is used to differentiate varieties that differ evenmarginally from each other. Polymerase Chain Reaction

(PCR)-based methods such as Random Amplified Polymor-phic DNA (RAPD) [24,5] can be a powerful tool for theauthentication of plant materials under study [15]. Therefore,

random amplified polymorphic DNA (RAPD) is the markerof choice in present study. A large number of reports have ap-peared in the literature using RAPD patterns for differentiat-

ing varieties, species, etc. of crop plants. These includestudies on mango (Mangifera indica) [22,13], pomegranates(Punicagranatum) [20], onion [18], pea [17], etc. wherein subtle

differences in the banding patterns have been used as an indexto differentiate varieties and assess genetic variability. Untilnow, to the best of our knowledge no finding has been reportedfor molecular marker based characterization of L. sativum.

Thus, the prime objective of the present study was to examinethe genetic diversity by employing RAPD.

2. Materials and methods

2.1. Plant material

Eighteen genotypes of L. sativum were used in the presentstudy for analysis of genetic diversity. Different genotypes

were obtained from Medicinal and Aromatic Plants Section,Chaudhary Charan Singh Haryana Agriculture University, Hi-sar. Seed samples were coded as (Hisar Chanderasur) HCS1-

18. Fresh leaf samples from 10 to 15 days grown plants weretaken for DNA extraction.

2.2. Genomic DNA extraction

The genomic DNA was extracted from 10 to 15 days old leavesof 18 genotypes of L. sativum by CTAB extraction method ofMurray and Thompson (1980), modified by Saghai-Maroof

et al. (1984) and Xu et al. (1994) with slight modifications. Fivegrams of fresh leaves were crushed using liquid nitrogen usingpre-sterilized mortar and pestle. The powder obtained was

thoroughly mixed with 10 ml of pre warmed extraction buffer(0.1 M Tris pH 8.0, 20 mM EDTA, 1.4 M NaCl, 1.5% CTAB).Just before the addition of extraction buffer to the crushed

material 0.2% b-mercaptoethanol was added to it. The mix-ture was incubated at 65 �C for 60 min with gentle mixing afterevery 15 min and centrifuged at 10,000 rpm for 10 min. In ob-tained supernatant equal volume of chloroform: isoamylalco-

hol mixture (24:1) was added and centrifuged at 10,000 rpmfor about 10 min at 4 �C. This step was performed thrice andto the obtained supernatant equal volume of chilled isopropa-

nol was added to precipitate DNA and was incubated at�20 �C overnight. The mixture was centrifuged at 7000 rpm

for about 10 min and the precipitated DNA was sterilized byadding 500 ll of 70% ethanol and centrifuged at 7000 rpmfor 5 min. The precipitated DNA was air dried and dissolved

in TE buffer (10 mM Tris pH 8, 1 mM EDTA pH 8) at roomtemperature and stored at �20 �C until further use. RNA freeDNA was obtained by the addition of 1 ll of RNAse (10 mg/

ml) to 100 ll of extracted DNA and incubated at 37 �C for30 min. The quality of RNase treated extracted DNA wasquantified by running it on 0.8% agarose gel containing

5 lg/ml of ethidium bromide against a known standardDNA using alpha imager software .

2.3. RAPD PCR amplification

The conditions to carry out amplification of L. sativum usingPCR were optimized. The best amplified products were ob-tained using 20 ll of reaction mixture which specifically con-

tained 50 ng template DNA, 0.5 lM primer, 0.5 mM of eachdNTP, 2.5 mM MgCl2, 1X Taq Polymerase buffer, 1 U TaqPolymerase. PCR amplification was performed to fulfill 30 cy-

cles after an initial denaturation at 94 �C for 5 min. Each cycleconsisted of a denaturation step at 94 �C for 1 min, an anneal-ing step at annealing temperature and extension at 72 �C for

2 min, following by extension cycle for 5 min at 72 �C in thefinal cycle. The various annealing temperatures of 36, 37, 38,39, and 40 �C produced variable banding patterns. A set of50 random decamer primers (Bangalore Genei, India) with

more than 50% GC content were selected out of which 32primers were selected for data analysis. Samples of 10 llRAPD–PCR product were analyzed by electrophoresis on

1.5% agarose gel in 1X TBE buffer. The sizes of DNA frag-ments were estimated by comparison with standard ladder(1 kb; Bangalore Genei, India) containing 5 lg/ml of Ethidium

bromide. Then the gels were visualized, photographed andanalysed.

Table

2Pairwisesimilarity

indices

of18genotypes

ofL.sativum

usingRAPD

analysis.

HCS-l

HCS-2

HCS-3

HCS-4

HCS-5

HCS-6

HCS-8

HCS-10

HCS-l1

HCS-12

HCS-13

HCS-14

HCS-15

HCS-16

HCS-17

HCS-l8

HCS-20

HCS-21

1HCS-1

1HCS-1

0.3763

1HCS-2

0.4579

0.5367

1HCS-3

0.3316

0.4737

0.5145

1HCS-4

0.1737

0.3764

0.3281

0.3824

1HCS-5

0.3204

0.4717

0.3943

0.3828

0.391

1HCS-6

0.4093

0.4608

0.4880

0.5484

0.3258

0.3846

1HCS-8

0.3148

0.4920

0.4029

0.4084

0.4086

0.4022

0.5072

1HCS-10

0.2857

0.3913

0.3503

0.4556

0.4059

0.3902

0.4748

0.4368

1HCS-11

0.2241

0.2919

0.3797

0.4245

0.3546

0.3636

0.3048

0.3719

0.3557

1HCS-12

0.2489

0.3284

0.3255

0.4185

0.3229

0.2462

0.3955

0.3521

0.4402

0.3152

1HCS-13

0.4082

0.2830

0.3158

0.3693

0.2423

0.2581

0.4462

0.3731

0.3917

0.2883

0.5650

1HCS-14

0.4046

0.3160

0.3712

0.4341

0.2161

0.2212

0.4783

0.3375

0.3796

0.3158

0.4558

0.5785

1HCS-15

0.3732

0.2963

0.3621

0.3993

0.2517

0.2102

0.4517

0.3535

0.3298

0.2634

0.3972

0.6076

0.6236

1HCS-16

0.4064

0.3333

0.3822

0.4083

0.2397

0.2214

0.4382

0.3225

0.2955

0.2564

0.3574

0.5053

0.5058

0.6585

1HCS-17

0.3290

0.3665

0.3684

0.4106

0.2838

0.2986

0.4261

0.4053

0.4028

0.3053

0.3348

0.3869

0.4892

0.4655

0.4697

1HCS-18

0.0845

0.1710

0.1859

0.3012

0.2667

0.1124

0.2417

0.2089

0.25

0.2555

0.2983

0.2614

0.2885

0.3120

0.2632

0.2372

1HCS-20

03951

0.4093

0.3871

0.3065

0.2335

0.2697

0.3962

0.4268

0.3016

0.2227

0.3042

0.4158

0.4297

0.4462

0.4894

0.5265

0.2065

1HCS-21

42 D. Bansal et al.

2.4. RAPD analysis

RAPD patterns were analysed by scoring presence (1) or ab-sence (0) of bands for estimation of similarity among all testedsamples. The matrix of similarity (Jaccard) and similarity of

coefficients [14] were calculated and the dendrogram obtainedby clustering according to the Unweighted Pair-Group Meth-od with Arithmetic averages (UPGMA) using NTSYS-pc soft-ware. Principal component analysis along with 2D and 3D

plots were constructed using eigen values and vectors.

3. Results and discussion

The RAPD technique had been successfully used in number oftaxonomic and genetic diversity studies [8,1]. RAPD markershave been used in this study for the assessment of genetic rela-

tionships among 18 genotypes of L. sativum. The choice ofRAPD technique was motivated by the fact that no DNA se-quence information is known about these species. Initially 50

decamer primers were screened, out of which only 32 primersshowed reproducible fragments with easily recordable bands.Total of 414 reproducible and clear bands were distinguished

across the selected primers and statistical analysis showed361 polymorphic bands and 53 monomorphic bands (Table 1)Similarly, extensive polymorphism was detected by Dharmarand De-Britto [7] with six primers in genotypes of Withania

somifera. Six primers generated reproducible, informativeand easily scorable RAPD profiles. These primers produced

Table 3 List of primers capable of amplifying unique alleles

from different genotypes of L. sativum.

S. No. Primer No. of unique

alleles

Allele size

(bp)

Genotypes

1 6800-024 2 1261 HCS-20

570 HCS-21

2 6800-031 2 542 HCS-1

465 HCS-1

3 6800-032 3 2091 HCS-3

800 HCS-17

600 HCS-14

4 6800-037 2 1750 HCS-20

540 HCS-18

5 6800-038 4 1192 HCS-21

600 HCS-18

567 HCS-21

310 HCS-21

6 6800-044 3 8000 HCS-13

7200 HCS-20

3750 HCS-14

7 6800-049 3 1000 HCS-21

750 HCS-20

400 HCS-17

8 6800-054 1 1318 HCS-3

9 6800-060 4 2833 HCS-17

1750 HCS-2

1375 HCS-21

1125 HCS-17

10 6800-063 3 667 HCS-18

443 HCS-18

400 HCS-18

Figure 2 Dendrogram (NTSYS-pc) constructed with UPGMA clustering method in Lepidium sativum using 32 primers. The scale in the

figure is genetic similarity coefficient calculated according to Jaccard’s.

Assessment of genetic diversity in Lepidium sativum (Chandrasur) a medicinal herb 43

multiple band profiles with a number of amplified DNA frag-

ments varying from 5 to 9.The highest level of polymorphism (94%) was produced by

five primers (6800-018, 6800-025, 6800-031, 6800-058 and

6800-060) and the least polymorphism (66.67%) was exhibitedby two primers (6800-017 and 6800-055). The mean percentageof polymorphic bands observed in L. sativum was

82.59 ± 1.68%. RAPD analysis of Commiphora wightii wascarried out by Suther et al. Suthar et al. [22] using a set of40 custom-made decamer random primers, of which 12 prim-ers responded. Out of 79 bands, 13 bands (16.5%) were mono-

morphic and remaining 66 bands (83.5%) were polymorphic.Arya et al. Arya et al. [2] reported the use of 12 random prim-ers for genetic diversity studies of Cassia occidentalis. 111

bands on RAPD profile were scored with 79 bands showingpolymorphism (71.17%). Nine out of twelve primers gavemore than 60% polymorphism. Fan et al. Fan et al. [9] re-

ported 97.9 % polymorphism in Ginkgo biloba using RAPDmarkers.

The amplified bands in 18 genotypes of L. sativum using 32

primers varied from 3 (6800–017) to 25 (6800–025) per primerwith an average of 12.93 ± 0.94 and an average of11.28 ± 0.91 polymorphic bands per primer (Fig. 1). Zouet al. Zou et al. [25] detected polymorphic fragments ranging

from 3 to 8 bands per primer and with an average of 5.48bands per primer in Curcuma. In RAPD profiling of C. wightii,a total of 79 reproducible bands were produced. The number

of bands produced per primer ranged from 4 to 11. A totalof ten primers detected in this study which produced twelveunique alleles in 18 genotypes (Table 3). These primers can

be utilized to distinguish one or a few genotypes from the rest

of genotypes. Borgohain et al. Borghohain et al. [4] and Carelliet al Carelli et al. [6] reported similar studies in Capsicum andLycopersicon esculentum.

The pairwise comparison of the RAPD profiles based onboth shared and unique amplification products were used togenerate similarity matrix. The similarity indices between dif-

ferent genotypes ranged from 0.085 (HCS-20 and HCS-1) to0.658 (HCS-16 and HCS-17) (Table 2). This infers that leastsimilarity was found in between HCS-1 and HCS-20 and max-imum similarity was found in between HCS-16 and HCS-17.

The average similarity among all the genotypes was found tobe 0.363. Binary RAPD data of L. sativum was used to pro-duce dendrogram using NTSYS-pc. Cluster analysis of the

genotypes based on UPGMA divided the 18 genotypes intotwo main clusters, with first cluster having only HCS-20 geno-type of L. sativum and other having rest of all 17 genotypes

(Fig. 2). The dendrogram based on similarity matrix revealed23–66% genetic relatedness among 18 genotypes. Cluster IIwas further divided into two subclusters at 0.34 similarity coef-

ficient. Subcluster I included HCS1, HCS14, HCS15, HCS 16and HCS 17. HCS 16 and HCS 17 seems to be more close toeach other as compared to others this may be due to its pedi-gree. In subcluster II HCS 12 seemed to be out grouped from

other genotypes of this group. Hepsibha et al. Hepsibha et al.[10] used NTSYS-pc for cluster analysis of Azima tetracanthagenotypes and on the basis of similarity matrix by UPGMA

genotypes were clustered into two main clusters. Cluster anal-ysis based on similarity values in Jatropha classified the Jatro-pha curcas population in two major clusters at a similarity

Figure 3 A two dimensional and three-dimensional plot of principal component analysis based on RAPD in Lepidium sativum

genotypes.

44 D. Bansal et al.

coefficient of 0.54, cluster I comprised JC-2 and JC-19 andcluster II comprised of 38 genotypes [3]. The principal compo-

nent analysis (PCA) apart from cluster analysis was performedfor grouping of accessions. Applying both methods was recom-mended to extract the maximum amount of information from

the matrix data [14]. Clustering was useful in detecting rela-tionships among genotypes, while PCA allowed a view onthe relationships between groups. HCS-17 is uniquely depicted

at PCA value of 0.11 (Dim-2) and 0.41 (Dim-1). Three mainclusters were depicted in the 2-D plot of PCA. First clusterfound at PCA values of 0.47 (Dim-2) to 0.70 (Dim-1) wasoccupied by HCS-12, HCS-13, HCS-14, HCS-15, HCS-16

and HCS-18. Second main cluster at PCA value of 0.06(Dim-2) to 0.73 (Dim-1) was occupied by the varieties HCS-2, HCS-3, HCS-4, HCS-8, HCS-9 and HCS-7. Genotype

HCS 17 was in cluster I in dendrogram occupied their uniqueposition i.e., at the periphery in 3-D scaling while others com-prised cluster II. The results of PCA analysis based on RAPD

largely corresponded with the result of the cluster analysis(Fig. 3). This is the first report of genetic diversity studies ofL. sativum using DNA based molecular markers. The RAPD

patterns obtained from our study can serve as a vital inputto the conventional method of varietal identification that reliessolely on morphological character. The powerful capability ofmolecular technique to distinguish closely related varieties

based on their RAPD patterns has been brought out by thisstudy.

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