Abstract: Background and Objectives: Aloe is a medicinally and economically important genus. Many Aloes seem an endangered species because of over-collection, destruction of plants and destroyed of natural habitats. The objectives of current study was to survey, collect and identification of some Aloe species and to analyze genetic variations between the collected Aloe species. Materials and Methods: Four Aloe species (A. armatissima, A. edentata, A. parvicoma and A. pseudorubroviolacea) and Agave americana (Asperagaceae) were used as plant materials for ecological and genetic studies. In RAPD and ISSR analysis 23 and 16 primers, respectively were screened. Results: Ecological study showed that the 4 species are endemic: 2 are endangered (A. edentata and A. parvicoma) and the others are not-endangered (A. armatissima and A. pseudorubroviolacea), while A. americana was introduced as ornamental species. Concerning RAPD, a total of 134 reproducible bands of them 131 bands are polymorphic ~ 97.65% polymorphism were produced, which ranged from 9 bands (primer OPC-04) to 18 (primer OPA-03) bands, with an average 13.4 bands/ primer, ranging from ~300-2500 bp. According to ISSR, 113 reproducible bands were totally yielded with an average 12.6 bands/primer, from ~180-1500 bp, of which 107 poly-morphic bands number (PBN) ~94.96% polymorphism ranged from 10 bands (primer UBC-818 and primer UBC-819) to 14 (primer UBC-814) with an average of 11.9 PB/primer. Conclusion: The results revealed high genetic variations between 4 bands Aloe species and A. americana species, which will be in concern for improvement, breeding and conservation programs.
INTRODUCTION
Many Aloes seem an endangered species because of over-collection of plants, destruction of plants in harvesting leaf exudates and destroyed of natural habitats1. To develop the medicinal values and to remove the gap among supply of plant material and demand, for sustainable use in future, it is very urgent to conserve this species. To develop effective conservation, breeding program and management strategies, since morpho-chemical characters are dependent on age and environment, it is important to use genetic analysis to characterize this economically and medicinally important genus1.
Genus Aloe is a flowering succulents consisting of over 500 known species including shrubs, perennials and trees2. The habitat of approximately 50 species is the Arabian Peninsula and almost 30 species are present in western and central African countries. Worldwide, Aloe genus plants such as A. vera, A. perryi, A. arborescens and A. ferox are using widely for their medicinal properties3.
Since ancient times, treatment of inflammatory conditions, gastrointestinal disorders and microbial infections were carried out using the Aloes. It has different uses such as cosmetic, beverage and food industries4.
Analysis of phytochemical showed that several of carbohydrate polymers (notably glucomannans) and other phenolic compounds with low molecular weight are contained in many Aloe species4. Diverse DNA based molecular characterizations have been evolved as methods for analysis of variation and to establish similarity among cultivars and species5. RAPD is widely used as it permits a fast and low cost assay with distinctive primers6. Although the speed and simplicity of RAPD technique, it's been efficiently used to evaluate genetic structure and similarity analysis7 and effectively applied to research of genetic variability in some genus such as Gossypium8, Eucalyptus9, Asparagus10 and Mangifera11.
ISSR technique, PCR based method; it is an inexpensive technique, convenience of use and high stage of reliability in reproducing consequences12,13. ISSRs have excessive reproducibility probable compared to RAPD primers (10 mers) because of using longer primers (16-25 mers) with high annealing temperature (45-60°C) leading to higher stringency. ISSR is used extensively and is approved as a method in genetic researches of both cultivated and wild plants13.
In Aloe species, RAPD, amplified fragment length polymorphism (AFLP) and ISSR markers have been applied to investigate genetic diversity between Aloe species. Genetic analysis between 3 species A. vera, A. arborescence and A. ferox were performed using RAPD marker14. Analysis of genetic diversity using RAPD and morphological markers in some Aloe species was performed by Nayanakantha et al.15. Diversity of genetic amongst accessions of Aloe collected from different ecological regions in India and Iran, was assessed using RAPD, ISSR and AFLP markers16,17. As mentioned above, the morpho-chemical characters and genetic analysis are very important tools for improvement, breeding and conservation program of economically and medicinally important Aloe species. Therefore the main objectives of the current research are (1) Surveying, collecting and identification of Aloe species from Taif region. (2) Analysis of the genetic variations between the collected Aloe species using RAPD and ISSR as molecular markers.
MATERIALS AND METHODS
Plant materials: This study was carried out, since May 2018-June 2019, in plant molecular genetic and tissue culture laboratory, Deanship of Scientific Research, Taif University. Four Aloe species (A. armatissima, A. edentata, A. parvicoma and A. pseudorubroviolacea) and A. americana (Asperagaceae) were collected from different localities 5 years ago at Taif highlands of Saudi Arabia, it were transplanted at Taif University Campus. The collected wild materials were identified according to Collentette18 and Chaudhary19 as shown in Table 1.
Extraction of DNA: For DNA extraction, 0.1 g of fresh leaves tissues was collected from A. americana, A. armatissima, A. edentata, A. parvicoma and A. pseudorubroviolacea (Table 1). DNA extraction, quantity and quality of DNA were performed according to Attia et al.20.
| Table 1: | Identification of 4 Aloe species A. armatissima, A. edentata, A. parvicoma and A. pseudorubroviolacea |
Random amplified polymorphic DNA (RAPD): Twenty three RAPD primers, which were synthesized by Macrogen Inc. Biotechnology Company-Seoul-South Korea, were screened. Preparation and PCR reactions for DNA amplification were applied according to Attia et al.20.
Inter simple sequence repeat (ISSR): Sixteen ISSR primers, from the University of British Columbia (UBC) series which were synthesized by Macrogen Inc. Biotechnology Company-Seoul-South Korea, were used. Amplification of DNA and evaluation of RAPD and ISSR products were carried out as described by Attia et al.21.
Statistical analysis: For RAPD and ISSR analysis, the manual scoring present (1) or absent (0) was used for high resolution band patterns, average linkage between species was used to produce cluster analysis and dendrogram for RAPD, ISSR analysis by Hierarchical Cluster Analysis using (IBM SPSS Statistics Version 20).
RESULTS AND DISCUSSION
Ecological distribution study: The genus Aloe in Saudi Arabia includes 24 species, 45.8% of them are endangered endemic species , 16.7% of them are endemic but non-endangered and 37.5% are near endemic which recorded in Yemen and Saudi Arabia, while one is belonged to tropical region (A. niebuhriana) and other once belonged to Sudano-Zambezian region (A. vera var. officinalis). All of these species were recorded in Sarawat mountains18,22. On the other hand, the 4 species in present study are endemic: 2 of them are endangered (A. edentata and A. parvicoma) and the 2 others are not-endangered (A. armatissima and A. pseudorubroviolacea)18,19, while A. americana was introduced as ornamental species (Table 1).
Genetic variation analysis
RAPD analysis: Based on RAPD analysis, 10 primers out of 23 primers evaluated, were produced reproducible, distinct and polymorphic bands (Table 2). About 134 reproducible bands were produced with an average 13.4 bands/primer, ranging from ~300-2500 bp. The representative RAPD profiles using primers OPB-01, OPD-02 and OPD-03 are shown in Fig. 1a and b, respectively.
Polymorphic bands were produced in all RAPD primers showing ~97.65% polymorphism between 4 Aloe species and A. americana. Whereas, mono-morphic bands number (MBN) were 3 bands. As shown in Fig. 1b, the profile of A. edentata (lane 8) generated with primer OPD-03, showed 8 bands while A. americana, A. parvicoma and A. pseudorubroviolacea (lanes 6, 9 and 10, respectively) showed 4 bands were different from the other profiles. All RAPD primers produced unique bands with most of 4 Aloe species and A. americana in this study. Das et al.23 reported that RAPD analysis between eleven A. vera populations, yielded 138 polymorphic bands (~87.34% polymorphism) of 158 total amplicons, which indicating a wide genetic variability between populations.
Bhaludra et al.17 reported that analysis of genetic variability of A. vera collected from different regions of Hyderabad, RAPD and ISSR analysis yielded 71.8 and 80.9% of polymorphism with 4.34 and 4.47 polymorphic bands/primer, respectively.
Rathore et al.24 checked genetic stability of sweet variety of A. vera in vitro propagated plantlets by RAPD and ISSR markers. Nayanakantha et al.15 studied genetic variation between A. vera accessions using RAPD markers.
Mehetre et al.25 demonstrated that bands that are produced in all individuals considered as mono-morphic bands. On the other hand, bands which are not resulted in all individuals (one or more) are poly-morphic and that which are recorded in at least one individual are unique bands.
| Table 2: | RAPD primers and amplified products by RAPD analysis of 4 Aloe species and Agave americana |
NTB: Number of total bands, RBS (bp): Range of band size (bp), MBN: Mono-morphic bands number, PBN: Poly-morphic bands number, PB: Polymorphic bands | |
| Table 3: | Similarity matrix between 4 Aloe species and Agave americana based on RAPD analysis |
| Fig. 1(a-b): | RAPD analysis of 4 Aloe species and Agave americana, (a) Lanes 1-5 indicate to PAPD profiles of 4 Aloe species and Agave americana using primer OPB-01 and (b) Lanes 1-5 indicate to PAPD profiles of 4 Aloe species and Agave americana using primer OPD-02 and lanes 6-10 indicate to RAPD profiles of 4 Aloe species and Agave americana using primer OPD-03 |
M: DNA marker 100 bp |
|
| Fig. 2: | Dendrogram showing genetic relationship between 4 Aloe species and A. americana based on RAPD analysis |
Three unique bands, Fig. 1a, (1800, 750 and 550 bp) were resulted in A. americana species (lane1) generated with OPB-01 primer. However, the same primer generated 2 unique bands 900 and 1900 bp with species A. edentata (lane 3) and A. pseudorubroviolacea (lane 5) respectively. As shown in Fig. 1b, 3 unique bands (1500, 900 and 700 bp) were found in A. americana species (lane1) generated with OPD-02 primer. Whereas, one unique band 1200 bp was resulted in A. parvicoma species (lane 4) generated with the same primer. Two unique bands 2000 and 450 were recorded in A. edentata (lane 8) and A. parvicoma (lane 9) respectively, were generated with OPD-03 primer.
Das et al.23 noticed that unique bands that present in population and were absent in others, could be used as population-specific diagnostic markers when it cloned, sequenced and converted into a locus-specific sequence characterized amplified region (SCAR), it could apply in future for identification of plants belonging to that population.
The similarity matrix Table 3, ranging from 19-62% indicate that there is a highly genetic variability between 4 Aloe species and A. americana investigated in current work. The cluster analysis based on the similarity showed that, 3 main clusters were resulted in the dendrogram according to the similarity matrix between 4 Aloe species and A. americana.
Cluster I consisted of 2 sub cluster, sub cluster 1 containing 2 species A. armatissima and A. edentata while sub cluster 2 containing A. parvicoma. Cluster 2 and 3 containing A. pseudorubroviolacea and A. americana, respectively (Fig. 2).
| Fig. 3: | ISSR analysis of Agave americana and 4 Aloe species |
M: DNA marker 100 bp, lanes 1-5 and lanes 6-10 indicate to ISSR profiles of A. americana and 4 Aloe species using primers UBC-813and UBC-814, respectively |
|
| Table 4: | ISSR primers and amplified products by ISSR analysis of 4 Aloe species and Agave americana |
NTB: Number of total bands, RBS (bp): Range of band size (bp), MBN: Mono-morphic bands number, PBN: Poly-morphic bands number, PB: Polymorphic bands | |
According to the results of similarity matrix and the dendrogram, the highest similarity 62% was among A. armatissima and A. edentata followed by 54 and 51% similarity of A. parvicoma with A. edentata respectively, the lowest similarity 19% was between A. americana and A. pseudorubroviolacea. Within four A. species, lowest similarity 30% was resulted between A. parvicoma and A. pseudorubroviolacea.
Bhaludra et al.17 found that RAPD analysis among twelve collected elite accessions of A. vera from different places of India showed 71.8% molecular polymorphism.
Rana and Kanwar26 reported that similarity coefficient value ranged from 62-91% in RAPD analysis between 24 genotypes of A. vera L. collected from different provinces of Himachal Pradesh, India.
ISSR analysis: Hogbin and Peakall27 reported that structure of species and phylogenetic considered as a results of interaction among different factors, such as evolution of species, geographical range, seed dispersal, mating method, gene flow and genetic drift.
In current work, out of 16 primers, 9 primers with high intensity and relatively high polymorphism bands were selected and used to amplify 4 A. species and A. americana (Table 4). According to analysis of ISSR, 113 reproducible bands were totally yielded with an average 12.6 bands/primer, from~180-1500 bp, of which poly-morphic bands number (PBN) 107 with an average of 11.9 PB/primer, on the other hand the mono-morphic bands number (MBN) were 6 bands, Fig. 3 showed representative ISSR profiles using UBC-813 and UBC-814 primers. The results indicated that the poly-morphic bands percentage (PB%) ranged from ~83.3% with an average 94.96%, indicating high genetic variability between four Aloe species and A. americana (Table 4).
According to ISSR analysis to evaluate genetic variation in twelve elite accessions of genus A. vera. Bhaludra et al.17 they found that 85 bands, 81% polymorphism, of 105 total amplified bands. In the study to assess the genetic variation in A. vera L. genotypes from different provinces of Himachal Pradesh, India. ISSR analysis showed that 21 polymorphic with an average 87.5% polymorphism of 24 a total amplified bands27.
| Table 5: | Similarity matrix between 4 Aloe species and Agave americana based on ISSR analysis |
| Fig. 4: | Dendrogram showing genetic relationship between 4 Aloe species and Agave americana based on ISSR analysis |
After ISSR the results showed that a lot of unique bands were generated with most of ISSR primers used in current study, as shown in Fig. 3, Two unique bands 900 and 290 bp were found in A. americana species (lane 1) generated with UBC-813 primer. Whereas, two unique bands 550 (positive band) and 300 pb (negative band) were recorded in A. parvicoma (lane 4) and A. pseudorubroviolacea (lane 5) respectively, were generated with the same primer. Bhaludra et al.17 they reported that several unique bands have been resulted by ISSR analysis to evaluate genetic variation in A. vera germplasm collected from different geographical places of India. The cluster analysis (Fig. 4) based on the similarity showed that; 3 main clusters were resulted in the dendrogram according to the similarity matrix between A. americana and four A. species, cluster I consisted of 2 sub clusters, sub cluster one containing 2 species A. armatissima and A. edentata while sub cluster 2 containing A. parvicoma. Cluster 2 and 3 containing A. pseudorubroviolacea and A. americana, respectively. According to the results of similarity matrix (Table 5) and the dendrogram (Fig. 4), the highest similarity 72% was among A. armatissima and A. edentata followed by 69 and 65% similarity of A. parvicoma with A. armatissima and A. edentata, respectively, the lowest similarity 35% was noticed between A. pseudorubroviolacea and A. americana. Within 4 A. species, lowest similarity 45% was resulted between A. parvicoma and A. pseudorubroviolacea. Rana and Kanwar26 demonstrated that similarity coefficient values ranged from 0.38-1 with ISSR primers that were used to assess the A. vera L. genotypes collected from different regions of Himachal Pradesh, India, these results are consistent with current results that showed that RAPD and ISSR analysis revealed high genetic variations between 4 Aloe species and A. americana species.
CONCLUSION
In conclusion, the results showed that RAPD and ISSR analysis revealed high genetic variations between 4 Aloe species and A. Americana species. These results will be applied for improvement, breeding and conservation programs of Aloe species in future.
SIGNIFICANCE STATEMENTS
The present study proved that it is important to use genetic analysis along with morphological characters to study and characterize Aloe species. These results will help us and other researchers to study the endangered and threatened medicinally and economically important wild plants especially in high altitude regions like Taif province.
ACKNOWLEDGMENT
Authors are very gratefully to "Vice-Presidency of Graduate Studies and Academic Research, University of Taif" for the financial support of this project (Ecological distribution and genetic variations of some Aloe species in Taif, KSA), the project number (1-438-5901).