MATERIALS AND METHODS

Collection of plant material: Twelve accessions of Aloe vera germplasm (NBPGR, New Delhi, India vide voucher no: NBPGR/2011/1771 dated 11.07.2011) representing totally from different geographical regions of India were obtained from Rajasthan (IC111267, IC111269, IC111271, IC111272), Gujarat (IC111279), Haryana (IC111280), Delhi (IC471882, IC471883, IC471884 and IC471885) and Central Institute of Medicinal and Aromatic Plants (CIMAP) Research Center, Boduppal, Hyderabad, Telangana State, India (Aloe CIM-Sheetal and commonly grown wild Aloe vera-local) vide voucher No: CIMAP/63/6222 dated 20.07. 2011. All the plants utilized in the present study were about two years of age. In all cases, the predominantly grown accession was used. In case of some states, where variation has been reported different provenances were collected. Accordingly, the accessions from the different regions of collection from Rajasthan, Gujarat, Haryana, Delhi, Uttarpradesh and Telangana State; the germplasm was maintained at the research farm of Indian Immunologicals Ltd, Hyderabad, India and the details of the accessions are presented in Table 1.

DNA extraction: Total genomic DNA was extracted from younger leaves of 12 Aloe vera accessions by following the standard CTAB method with minor modifications (Doyle and Doyle, 1990). Two grams of leaves were ground in liquid nitrogen, then homogenized in 10 mL of extraction buffer (4% CTAB, 20 mM EDTA, 2% PVP, 1.4 M NaCl, 100 mM Tris-HCl pH 8.0 and 1% β mercapto ethanol) and incubated at 65°C for 30 min. The supernatant was twice extracted with chloroform: isoamylalcohol (24:1, v/v) and treated with 2 μL RNase A (100 mg/mL), incubated at 37°C for 1 h. The DNA was precipitated with isopropanol and washed twice with 70% ethanol. The pelleted DNA was air dried and re suspended in 500 μL of sterile Millipore water and finally all DNA samples were diluted to get 50 ng/μL and were stored at -20°C for use in RAPD and ISSR assay (Table 2).

RAPD analysis: A set of 64 decamer primers (Table 3) synthesized from Integrated and Technologies (IDT) were used for DNA amplification with minor modifications (Williams et al., 1990). The PCR amplification was performed in 10 μL reaction volume containing of 5 ng of genomic DNA, 1x PCR buffer (10 mM Tris pH 9.0, 50 mM KCl, 1.5 mM MgCl₂), 100 μM of each the 4 dNTPs, 0.4 μM of RAPD primer and 0.3 U of Taq DNA polymerase (Bangalore Genei). PCR amplifications were performed in Gene Amp 9700 Thermal Cycler (Perkin Elmer Applied Biosystems) with an initial denaturation at 94°C for 3 min followed by 45 cycles of 94°C for 45 sec, 36°C for 30 sec and 72°C for 2 min with a final extension of 72°C for 7 min.

Table 1:	Details of Aloe vera accessions used in diversity analysis

Table 2:	Jaccard’s similarity coefficient values of 12 Aloe vera accessions based on pooled data of RAPD and ISSR primers

Table 3:	List of RAPD primers

The PCR amplified products were separated on 1.5% agarose gel in 1x TAE buffer by electrophoresis at 100 V for 2 h and visualized with ethidium bromide staining and the gels were documented using an Alpha Image Gel Documentation System.

ISSR analysis: A total of twenty five ISSR primers (UBC primer set No. 9) were used for the analysis (Table 4). The PCR reaction mixture (10 μL) consisted of 5 ng of genomic DNA, 1x PCR buffer (10 mM Tris pH 9.0, 50 mM KCl, 1.5 mM MgCl₂), 200 μM of each the four dNTPs, 0.4 μM of ISSR primer and 0.3 U of Taq DNA polymerase (Bangalore Genei). PCR amplifications were performed using a GeneAmp 9700 Thermal Cycler (Perkin Elmer Applied Biosystems) with initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 30 sec, 1 min at the annealing temprature (Ta), 72°C for 2 min with a final extension of 72°C for 7 min. The amplified products were electrophoresed at 100 V on a 1.7% agarose gel using EcoRI and HindIII double digest as the molecular weight standard.

Statistical analysis: For each RAPD and ISSR primer, the presence or absence of bands in each accession was visually scored and set in a binary matrix. The number of polymorphic and monomorphic fragments for each primer pair was scored and the monomorphic markers were excluded from the analysis.

Table 4:	List of ISSR primers

The binary matrices were read by NTSYS-pc version 2.02i (Rohlf, 1997) with Jaccard’s similarity coefficients and estimates of genetic distances for all pair wise comparisons between accessions were determined using Similarity for Qualitative Data (SIMQUAL). Dendrograms were created independently for both the marker systems and also based on pooled marker data using Unweighted Pair Group Method with Arithmetical Averages (UPGMA). The correlation between matrices was determined using Mantel test. Principal coordinate analysis was also performedand the ordination displayed in two dimensions.

RESULTS

Both the marker systems being employed to assess the genetic diversity in Aloe vera accessions were quite informative and were able to generate adequate polymorphism (Fig. 1a, b) among the Aloe vera accessions tested.

RAPD analysis: Out of 64 RAPD primers tested, 61 primers produced amplification products, of which 58 revealed polymorphic fingerprint patterns. The number of bands amplified per primer varied from 4 (OPA-5) to 17 (OPA-3). A total of 351 bands were amplified, of which 252 were observed to be polymorphic resulting in a polymorphism frequency of 71.8% and average of 4.34 polymorphic bands/primer.

Fig. 1(a-b):	(a) RAPD and (b) ISSR profile of 12 Aloe vera accession amplified with OPA-09 and UBC-861 Primer. Lanes 1-12 the samples used in the study as listed in Table 1 and lane M represents λ DNA double digest with EcoRI and Hind III restriction enzymes. Arrows indicates the accession specific bands

The molecular size of the bands varied between 100-2600 bp and several accession specific bands have been identified (Fig. 1a). The extent of polymorphism per primer ranged from with ten alleles per primer OPA-9 (0.98%) to OPA-17 (0.87%) with three alleles per primer. The similarity matrix values using Jaccard’s coefficient based on RAPD markers ranged from 0.082 between IC111272 and IC111269 to 0.890 between accessions IC111280 and IC111279. At 17% similarity, the accessions separated out into two major clusters and clustering of accessions based on dendrogram and PCO analysis was similar in pattern.

ISSR analysis: Of the 25 ISSR primers screened, 24 primers produced amplification products, of which 19 primers revealed polymorphic loci across the Aloe vera accessions tested. A total of 105 bands were amplified of which 85 amplicons were observed to be polymorphic resulting in polymorphism of 80.9%. The average number of polymorphic amplicons per primer was 4.47%. The number of bands amplified per primer varied between 2 (UBC primer No. 860) and 14 (UBC primer No. 867).

Fig. 2:	Dendogram (UPGMA) representing genetic relationships among 12 accessions of Aloe vera based on Jaccard’s similarity coefficients obtained using the pooled allelic profile of RAPD+ISSR primers

The size of the amplicons varied between 100-2800 bp and several accession specific bands have been identified as in Fig. 1b. The extent of polymorphism varied with ten alleles between UBC 867 (0.98%) to UBC 861 (0.82%) with two alleles per primer. Similarity matrix values based on ISSR markers ranged from 0.171 between IC111280 and IC111267 to 0.745 between accessions IC471883 and IC471882. Dendrogram analysis separated the accessions into two clusters at 29% similarity. Clustering of accessions based on dendrogram and PCO analysis was similar for most accessions except for the accessions IC111269 which is separately sub-grouped in cluster II.

Combined RAPD and ISSR analysis: The genetic similarity matrix data generated using RAPD and ISSR systems were compared. Mantel test for congruence of RAPD and ISSR data matrices indicated a goodness of fit (r = 0.87158) indicating good correlation between the two molecular marker systems. Although the two marker systems sampled different segments of the genome, the clustering pattern of the genotypes was almost similar with both the marker systems and most of the accessions were placed in their respective clusters with minor changes.

The genetic relatedness of the accessions was determined using Jaccard’s similarity coefficients. Cluster analysis (UPGMA) performed from combining polymorphic data of both markers (RAPD and ISSR) generated a dendrogram that separated the accessions into two major clusters at 48% of variation (Fig. 2). The first cluster (I) comprised IC111267, IC111271 and IC111272. The second cluster (II) further divided into 2 sub-clusters. Among them the first sub-cluster (IIa) comprised of IC111269, IC111279, IC111280, IC 471885, wild Aloe vera and Aloe CIM-Sheetal (CAL14) and the second sub-cluster (IIb) comprised of IC471882, IC471883 and IC471884. The accession IC111280 and IC111279 appeared to be closer to each other with a similarity coefficient of 0.847 (Table 2), while the divergent accessions were IC111279 and IC111272 (similarity coefficient 0.143). Principal Coordinate Analysis (PCO) based on genetic similarity showed the relationship among accessions in two dimensional spaces. The PCO analysis based on pooled data of RAPD and ISSR primers grouped the accessions into three groups as given in Fig. 3 which is similar to the UPGMA clustering pattern.

Fig. 3:	Two-dimensional scaling of Aloe vera accessions by principal component analysis using pooled data of RAPD and ISSR primers

DISCUSSION

An understanding of the extent of genetic diversity is important for both plant breeding and germplasm collection. In Aloe vera traditional methods like horticultural traits are relatively less reliable and inefficient for precise discrimination of closely related genotypes (Nejatzadeh-Barandozi et al., 2012). Hence, selection based on genetic information using neutral molecular markers is essential as it is more reliable and consistent. Among different marker systems available, RAPD and ISSR markers became popular in diversity studies because of simplicity, rapid, inexpensive and applicable to any genome without any prior information regarding the genome of the plant.

In the present study, a set of 12 elite Aloe vera accessions (Table 1) were analyzed using 64 RAPD and 25 ISSR markers to describe the genetic structure among the accessions. The RAPD primers revealed 71.8% polymorphism with 4.34 polymorphic bands/primer, while ISSR primers revealed 80.9% polymorphism with 4.47 polymorphic bands/primer indicating wide genetic variation among the accessions. ISSR primers detect more polymorphism than RAPD primers because of variability in microsatellite loci due to DNA slippage (Williams et al., 1990). The RAPD markers cover the entire genome in coding and non coding regions including repeated or single-copy sequences, while ISSR markers disclose polymorphism from sequences between two microsatellite primer sites (Williams et al., 1990; Zietkiewicz et al., 1994). The ISSR method has been reported to be more reproducible (Goulao and Oliveira, 2001) and produces more complex marker patterns than the RAPD approach (Chowdhury et al., 2002; Parsons et al., 1997) reported that is an advantageous when differentiating closely related cultivars. Both the marker techniques provides a useful approach for evaluating genetic differentiation, significantly in those species that are poorly known genetically and are propagated vegetatively like monocot genus in Musa (Bhat and Jarret, 1995) and Lilium (Haruki et al., 1998).

The PCO analysis and dendrogram constructed based on RAPD+ISSR polymorphism showed similar clustering pattern and disclosed predominantly two major clusters (I and II). The similarity values ranging from 14 to 85% indicating that there is a remarkable genetic variation among Aloe vera accessions used in the present study. The highest similarity (85%) was recorded between IC111280 and IC111279 accessions followed by 82% similarity between wild Aloe vera and Aloe CIM-Sheetal accessions which were collected from CIMAP Research Center, Hyderabad, India. Recently, AFLP based characterization of Aloe vera accessions from different location of Madhya Pradesh, India has reported modest level of genetic variability (Tripathi et al., 2011). This low level of variability could be because of small sample size collected from the limited geographical regions. Similarly, Darokar et al. (2003) has also reported the morphological similarity 78.8-99% in Aloe vera accessions revealed by RAPD and AFLP analysis. Nayanakantha et al. (2010) have reported a good amount of genetic variability among Aloe vera accessions based on RAPD analysis.

CONCLUSION

Apparently, the present study constitutes the presence of wide genetic variability among the Aloe vera accessions obtained from NBPGR, New Delhi, India. This variability can be used for genetic improvement through breeding programs and the accession specific bands were identified in this study will provide tags for future genetic improvement as well as in authenticating the genotypes. Both the markers techniques (RAPD and ISSR) have been shown to be useful in detecting small genetic variations within and among Aloe vera populations.

ACKNOWLEDGMENT

The authors profusely thank Department of Genetics and Biotechnology, Osmania University, Hyderabad, India, for funding the research work. Germplasm was arranged by Dr.Vandana Tyagi, Principal Scientist, ICAR- National Bureau of plant Genetic Resources (NBPGR), New Delhi, India for this study.