Amandeep Kaur?Rakesh Kumar?Suman Rani?Anita Grewal

Genetic diversity analysis of Lepidium sativum(Chandrasur)using inter simple sequence repeat(ISSR)markers

Amandeep Kaur?Rakesh Kumar?Suman Rani?Anita Grewal

Lepidium sativum(commonly known as garden cress)belongs to the family Brassicaceae.It is a fastgrowing erect,annual herbaceous plant.Its seeds possess significant fracture healing,anti-asthmatic,anti-diabetic, hypoglycemic,nephrocurative and nephroprotective activities.In the presentstudy,we assessed the genetic diversity of various genotypes of L.sativum using inter-simple sequence repeat(ISSR)markers.Out of 41 ISSR primers screened,32 primers showed significant,clear and reproducible bands.A totalof510 amplifi ed bands were obtained using 32 ISSR primers,outof which 422 bands were polymorphic and 88 bands were monomorphic.The percentage of polymorphism was found to be 82.A totalof 35 unique alleles ranging insize from 200 to 2,900 bp were observed. Cluster analysis based on unweighted pair-group method, arithmetic mean divided the 18 genotypes into two main clusters,with the first having only HCS-08 genotype of L. sativum and other having allof the other 17 genotypes.The Jaccard similarity coefficient revealed a broad range 32–72%genetic relatedness among the 18 genotypes.

ISSRGenetic diversityPolymorphismLepidium sativumCluster analysis

Introduction

Lepidium sativum L.,known as‘‘Chandrasur’’in Ayurveda,is one ofthe mostcommonly used traditionalmedicines in India and is mainly used to treat bronchial asthma, hiccups,cough with expectoration and bleeding piles (Pande et al.2002).The plant,considered to have originated in the highland regions of Ethiopia and Eritrea,is also known for its long-term use in fracture healing and rheumatic joints to relieve the pain and swelling(Juma 2007).

Lepidium sativum(Garden Cress)is an annual erect herbaceous plant,growing up to 30 cm,which belongs to family Cruciferae(Brassicaceae).The leaves of this plant are antiscorbutic,gently stimulant,diuretic and used to treatliver diseases(Paranjape and Mehta 2006).The seeds of L.sativum are tonic,diuretic,demulcent,aphrodisiac, carminative,rubefacient,galactogogue,emmenagogue and laxative(Yadav et al.2010).The seeds are applied as a poultice for wounds and sprains(Mali et al.2007)and as mucilage for dysentery and diarrhoea.The seeds are also a rich source ofthe rare group of imidazole alkaloids known as lepidine(Maier et al.1998)which are also used in the treatment of bacterial and fungal infections(Mukhopadhyay et al.2010).The gum produced from this plant has high molecular weight.It has various characteristics like binding,disintegrating,and gelling.(Dharmar and De-Britto 2011).Due to its medicinalproperties lepidium is in great demand by pharmaceutical industries.

A thorough understanding of the distribution of genetic variation at the molecular level in L.Sativum is essential for the conservation and authentication of its germplasm. The genetic structure of plant populations reflects the interactions of various processes including the evolutionary history of the species,mutation,genetic drift,matingsystem,gene flow,and selection.Recent developments in molecular techniques provide the scientist with a larger array of genetic tools for studying genetic diversity within and among populations.A large number of polymorphic markers are required to measure genetic relationships and genetic diversity in a reliable manner(Souframanien and Gopalakrishna 2004).This limits the use of morphological characters and isozymes,which are a few.

Molecular markers have developed into a powerful tool to analyze genetic relationships and genetic diversity.Intersimple sequence repeats(ISSRs)are the mostwidely applied because they do not require the knowledge of genomic sequences and are also simple,rapid,and cost effective. ISSR is a novelPCR technique thatuses repeat-anchored or non-anchored primers to amplify DNA sequences between two inverted SSRs(Zietkiewicz et al.1994).Several properties ofmicrosatellites such as high variability among taxa, ubiquitous occurrence,and high copy number in eukaryotic genomes make ISSRs extremely useful markers.They exhibitspecificity ofsequence-tagged-site markers,butneed no sequence information for primer synthesis,enjoying the advantage of random markers.

ISSR markers have been successfully used for the assessmentof genetic diversity ofvarious medicinalplants like Tribulus terrestris(Sarwat et al.2008),Papaver somniferum L.(Acharya and Sharma 2009),Anoectochilus formosanus(Zhang et al.2010),and Rheum officinale (Wang et al.2012).This study marks the first report on genetic diversity in L.Sativum using ISSR markers.Our a findings will aid in the long-term identification of diverse parental lines to generate segregating populations for tagging important traits with molecular markers.

Materials and methods

DNA extraction,purification and ISSR-PCR amplification

Leaves from 10 to 15 day-old plants were used for the genomic DNA extraction of18 genotypes of L.sativum,by using slight modified CTAB extraction method of Murray and Thompson(1980),modified by Saghai-Maroof et al. (1984)and Xu et al.(2004)and dissolved in TE buffer [10 mM Tris pH 8.0,1 mM EDTA pH 8.0]and stored at -20C until further use.DNA quantifications were performed by UV-spectrophotometer at 260 and 280 nm and the purity was then determined by calculating the ratio of absorbance at 260 nm to that of 280 nm(OD260/OD280). DNA concentration and purity were also determined by electrophoresis on 0.8%agarose gelcontaining 5 l g/mlof ethidium bromide against a known standard DNA using alpha imager software.The re-suspended DNA was then diluted in sterile distilled water to the concentration of 1 l g/l l for further use.

Out of 41 ISSR primers(Bangalore Genei,India,32 of them yielded bright and discernible bands and were selected for amplification(Table 1).The conditions to carry out amplification of L.sativum using PCR were optimized.ISSR amplification was performed using 20 l l of PCR reaction mixture containing 20 l lcontaining 50 ng of genomic DNA,1.0 l M primer,1.5 mMMgCl2,19 Taq buffer,1.5 U Taq DNA polymerase and 0.25 mM of each dNTPs(Bangalore Genei,India).Polymerase chain reaction(PCR)amplification was performed in a MyGeneTMSeries Peltier Thermal Cycler(Model MG96G)under the following conditions:initial denaturation of 5 min at 94C,followed by 30 cycles of 1 min denaturation at 94C,2 min annealing at45–52C and 1 min extension at 72C,ending with a final extension of 5 min at 72C.

PCR products were subjected to electrophoresis on 1.5%agarose gels buffered with 1X TBE detected by staining with ethidium bromide(0.5 l g/ml).The electrophoretic patterns of the PCR products were recorded digitally using an Alpha ImagerTMEC(Alpha Innotech)Gel documentation system.

Data analysis

For the genetic diversity analysis of ISSR markers,DNA bands were scored as present(1)or absent(0)to produce a setof binary data.Due to the dominance of ISSR markers, it was assumed that each DNA fragment position corresponds to an ISSR locus with two alleles revealed by band absence orpresence.The Jaccard similarity matrix of ISSR genotyping patterns was generated using this binary data. The resultantsimilarity matrix was employed to constructa dendrogram UPGMA,using an online software‘‘DendroUPGMA’’(Garcia-Vallve et al.1999).Principal componentanalysis was done to construct3D plots using Eigen values and vectors.

Results

Genetic diversity

Previous reports of molecular marker based genetic diversity in L.Sativum are a few.They were mainly based on morphological parameters,quantitative characters for yield components,fatty acid composition,and DNA fingerprint(Ottai et al.2012).In this study,ISSR markers have been used for the assessment of genetic diversity among 18 genotypes of L.sativum.Initially 41 ISSR primers were screened,out of which only 32 primersshowed significant,clear and reproducible bands.A totalof 510 amplified bands were distinguished across the selected primers and statistical analysis showed 422 polymorphic bands and 88 monomorphic bands(Table 1).The highest level of polymorphism(100%)was produced by seven primers:9973-008,9973-011,9974-057,9974-062, 9974-063,KJ-22,and KJ-25.

Table 1 List of primers used for ISSR amplification,amplified bands,percent polymorphism,melting temp

The least polymorphism(23.81%)was exhibited by primer 9974-056.The mean percentage of polymorphic bands observed in L.sativum was 82.00±21.82%.The amplified bands in 18 genotypes of L.sativum(using 32 primers)varied from 3(9974-057)to 28(9973-005)per primer,with an average of15.94±5.48 and an average of 13.18±5.88 polymorphic bands per primer(Fig.1).The size of all bands ranged from 200 to 2900 bp and the number of bands for each primer was from 11 to 19,with an average of 14.54 per primer.A total of 17 amplified primers produced 35 unique alleles in 18 genotypes (Table 2).The number of unique alleles ranged from 1 to 3 (Fig.2).These primers can be utilized to distinguish one or a few genotypes from the rest.

Cluster analysis

The pairwise comparison of the ISSR profiles,based on both shared and unique amplification products,were usedto generate similarity matrices.The similarity indices between different genotypes ranged from 0.324(HCS-10 and HCS-08)to 0.72(HCS-16 and HCS-15)(Table 3). This infers thatleastsimilarity was found in between HCS-08 and HCS-10 and maximum similarity was found between HCS-15 and HCS-16.The average similarity among all the genotypes was found to be 0.6.

Binary ISSR data of L.sativum was used to produce a dendrogram using UPGMA(Garcia-Vallve et al.1999). Cluster analysis of the genotypes based on UPGMA divided the 18 genotypes into two main clusters,with the first cluster having only HCS-08 genotype of L.sativum and the second having the 17 genotypes(Fig.3).The dendrogram based on a similarity matrix revealed 43–76%genetic relatedness among 18 genotypes.The second cluster was further divided into two sub-clusters at 0.48 similarity coefficient.Sub cluster I,which diverged at similarity coefficientof 0.52,was occupied by two genotypes,HCS-05 and HCS-21.Clustering was useful in detecting relationships among genotypes,while PCA allowed a view on the relationships between groups.The results of 3D PCA analysis based on ISSR support the cluster analysis of the dendrogram(Fig.4).

Discussion

Fig.1 ISSR-PCR fingerprints of 18 genotypes of L.sativum(HCS-01,HCS-02,HCS-03,HCS-04,HCS-05,HCS-06,HCS-08,HCS-10, HCS-11,HCS-12,HCS-13,HCS-14,HCS-15,HCS-16,HCS-17, HCS-18,HCS-20 and HCS-21)with primer BH-11.Lane M indicates Quantum PCR low range marker.The bands marked with arrows are the unique bands as detailed in Table 3

Table 2 List of primers capable of amplifying unique alleles from different genotypes of L.sativum using ISSR primers

The ISSR technique can detect more genetic loci than isozymes and has higher stability than RAPD.Our work is the first application of this method to L.sativum.The experimental results of the present study provide evidence for the reliability and usefulness of ISSR markers;those used here yielded reproducible polymorphic bands in 18 genotypes of L.sativum.A total of 510 amplified bands were distinguished across the selected primers,out of which 422 were polymorphic and 88 were monomorphic bands.

Fig.2 ISSR-PCR fingerprints of 18 genotypes of L.sativum(HCS-01,HCS-02,HCS-03,HCS-04,HCS-05,HCS-06,HCS-08,HCS-10, HCS-11,HCS-12,HCS-13,HCS-14,HCS-15,HCS-16,HCS-17, HCS-18,HCS-20 and HCS-21)with primer KJ-20.Lane M indicates Quantum PCR low range marker.The bands marked with arrows are the unique bands as detailed in Table 3

Fig.3 Cluster analysis of cumulative ISSR data in L.sativum.The Dendrogram was generated by the UPGMA clustering method.The scale on the bottom and the genotypes are indicated to the right of the dendrogram

The choice of ISSR technique was motivated by the fact that ISSR markers do not require a prior knowledge of the SSR target sequences,are highly reproducible due to their primer length and the high stringency achieved by the annealing temperature.They were also found to provide highly polymorphic fingerprints within and among species. Compared with the previously published RAPD-based (Bansal et al.2012)report,we revealed different diversity pattern of the L.Sativum:this may be due to reproducibility and utilization of highly polymorphic ISSR markers used in the present study.

Fig.4 3-D Principal component analysis using ISSR data of 18 L. sativum genotypes

ISSR markers were employed for genetic diversity analysis of cultivated ginseng,which revealed higher species level diversity in cultivated ginseng than its wild conspecifics(Lietal.2011).In fact,the ISSRs have a high capacity to reveal polymorphism and offer great potential to determine intra-and intergenomic diversity ascompared to other arbitrary primers like RAPDs.The highest level of polymorphism(100%)was produced by seven primers: 9973-008,9973-011,9974-057,9974-062,9974-063,KJ-22 and KJ-25 and the least polymorphism(23.81%)was exhibited by primer 9974-056.

Where as Kumar et al.(2011)reported 86.1%of polymorphism in genetic diversity analysis of Artemisia annua.About 17 primers produced 85 bands,of which 78 were polymorphic,with an average of 4.58 polymorphic fragments perprimer.The amplified bands in 18 genotypes of L.sativum using 32 primers varied from 3(9974-057)to 28(9973-005)per primer with an average of 15.94±5.48 and an average of 13.18±5.88 polymorphic bands per primer.Wang et al.(2012)had chosen thirteen primers to amplify a total of 199 individuals of 12 populations of R. officinale with 95.24%polymorphism.The size of all bands ranged from 200 to 2900 bp and the number ofbands by each primer was from 11 to 19,with an average of 14.54 per primer.

The similarity indices between different genotypes ranged from 0.324(HCS-10 and HCS-08)to 0.72(HCS-16 and HCS-15).This infers that least similarity was found in between HCS-08 and HCS-10,and maximum similarity was found in between HCS-15 and HCS-16.The average similarity among all the genotypes was found to be 0.6. The clusteranalysis ofthese genotypes,based on UPGMA, divided the 18 genotypes into two main clusters,with first cluster having only HCS-08 genotype of L.sativum and other having rest of all 17 genotypes.The dendrogram based on similarity matrix revealed 43–76%genetic relatedness among 18 genotypes.

Singh et al.(2012)conducted a genetic diversity relationship study in wild species of Brassicaceae and allied genera and obtained dendrogram by UPGMA analysis.The estimated similarity coefficients,using the Jaccard index amongst the eleven species ranged between 0.133(L.sativum/D.tenuisiliqua)and 0.785(D.tenuisiliqua/D.assurgens).The Group Iwas represented by the tribe Brassiceae while Group II contained members of two closely related tribes Lepidieae and Camelineae.

Similarly,Korkovelos et al.(Korkovelos et al.2008) reported the genetic similarity among eight Actinidia species calculated with Jaccard similarity coefficients from 0.100 to 0.579,indicating a broad range of variation from 0.100 to 0.579 and the eightspecies were clustered in two main groups and each main group in two subgroups.This indicates a close genetic relationship among Actinidia chinensis and Actinidia deliciosa species.Kumar et al. (2011)analysed a dendrogram based on UPGMA method utilizing ISSR markers thatgrouped allthe 20 genotypes of Artemisia annua collected from the Trans-Himalyan region into two main clusters with Jaccard’s similarity coefficient ranging from 0.57 to 0.81,where cluster Irepresents allthe genotypes from the Lee valley while cluster IIcontains all the genotypes from Nubra valley.The efficiency of this molecular marker for genotype identification in other medicinal plants has been reported previously and our results are consistent with these findings(Rahimmalek et al.2009;Farajpour etal.2012).

Plantgermplasm is wellrecognized as one ofthe crucial components for developing new crops and cultivars,while knowledge of its genetic variability is essential to its effective conservation and sustainable utilization.This is the first report of genetic diversity studies of L.sativum using ISSR markers.This study provides evidence that ISSR polymorphism could be used as efficient tool for the detection of similarities and phylogenetic relationships between the studied genotypes.

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11 March 2013/Accepted:21 December 2013/Published online:8 January 2015

The online version is available athttp://www.Link.springer.com

Corresponding editor:Chai Ruihai

Department of Biotechnology,University Institute of Engineering&Technology,Kurukshetra University, Kurukshetra 136119,Haryana,India

e-mail:anitapunia_17@rediffmail.com