Analysis of Genetic Fidelity of Wild Type and in Vitro Regenerated Aloe Vera Plants Through
RAPD and ISSR Molecular Markers
Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) marker assays were employed to investigate
polymorphism level of conventionally grown wild type and in vitro propagated Aloe vera plants. Despite having phenotypic similarities
in the plantlets, variation in the genomic constituents has been effectively established through RAPD and ISSR markers showing 33%
and 25% polymorphism respectively.
Aloe Vera, Wild type, in Vitro Regenerated, Genetic Fidelity, RAPD, ISSR.
Plant tissue culture techniques are known to induce somaclonal
variations. Frequency of these variations differ with the
source of explants, their regeneration methods, composition of
culture medium and cultural conditions. The first observation of
somaclonal variations was reported by Brown in 1984. Advances
in molecular biology have revolutionized every field of biological
sciences such as, DNA based markers have been used for individual
identification, genome mapping, pedigree and phylogenetic diversity
analysis in numerous taxa and in selecting somaclonal variations.
Molecular biological tools can accelerate artificial breeding
processes and clarify the genetic mechanisms that cannot be easily
detected with plant breeding techniques (Gupta et al., 2010). Molecular
tools can also give important information about the genetic distances between species (Mihalte et al., 2011).
The molecular marker technique efficiency is based on
the amount of polymorphism, it can detect in the given accessions
(Tharachand et al., 2012). Molecular marker studies on Aloe vera
have been published by using the following molecular techniques
such as: AFLP (Amplified Fragment Length Polymorphism),
RAPD (Random Amplified Polymorphic DNA), and ISSR (Inter Simple Sequence Repeats), (Alagukannan & Ganesh, 2016; Kumar
The increasing development and generalized use of
a large number of methodologies during the last years, requires
comparative studies in order to choose the best DNA marker technology
to be used in fingerprinting and in diversity studies, considering
reproducibility, costs, sensibility and level of polymorphisms
detection. Molecular technique comparisons have become important
because, depending on the objective of the study, one technique
can be more appropriate than another, as well as different
techniques being informative at different taxonomic levels. There
is a range of molecular marker types available, but the choice of
an appropriate genetic marker depends on the experience and competence
of the researchers, also on laboratory facilities. Several
works report comparable results among different markers while
others show considerable variations (Goulao & Oliveira, 2001).
Random Amplified Polymorphic DNA (RAPD)
Molecular markers have overcome limitations of morphological
and biochemical markers due to the influence of environment
on the performance of genotypes. A wide range of molecular
markers have been used to assess genetic diversity of Aloe vera
(Nayanakantha et al., 2010; Alagukannan & Ganesh, 2016). Random
Amplified Polymorphic DNA (RAPD) is powerful technique
considered as a useful tool, it need tiny amounts of DNA to give
rapid and accurate identification of alien species especially in the
developing countries, where DNA based methods are unavailable
due to their high cost, the requirement for complex equipments
and expertise (El-Mergawy et al., 2011). Among molecular markers
RAPDs are the most widely applied most probably because
they do not require the knowledge of genomic sequences and also
the protocol is relatively simple, rapid and cost effective (Bornet et
Inter Simple Sequence Repeats (ISSR)
The Inter Simple Sequence Repeats (ISSR) was first developed
by Zietkiewicz et al., (1994) to rapidly differentiate between
closely related individuals. This technique is known with
other names such as, MP-PCR (microsatellite-primed PCR) or ISA
(Inter-SSR Amplification) or RAMP (Randomly Amplified Microsatellite
Amplification) marker system. ISSR technique is also a
very simple, fast, cost-effective, highly discriminative and reliable
(Kumar et al., 2016).
The ISSR combines the advantages of AFLP markers and
SSR with the convenience of RAPD. It requires very small amount
of template DNA and is convenient in result recording and highly
reproducible. ISSR markers are known to be more reproducible
than RAPD markers and they have been successfully applied to
the study of genetic diversity in plants (Bornet et al., 2002). First,
it permits detection of polymorphisms in microsatellites and intermicrosatellites
loci without previous knowledge of the DNA
sequence. Microsatellite regions are abundant throughout the eukaryotic
genome, which are highly polymorphic in length and are
interspersed. Secondly, ISSR is informative about many loci and
are suitable to discriminate closely related genotype variants. And
lastly, ISSR markers constitute discrete markers suitable in the
DNA fingerprinting (El-Azeem et al., 2016; Wang et al., 2017).
Genetic diversity is the basis of plant breeding, so understanding
and assessing it is important for crop management, crop
improvement by selection, use of crop germplasm, detection of
genome structure, and transfer of desirable traits to other plants
(Sohrabi et al., 2012, Williams et al., 1990, Welsh & McClelland,
Materials And Methods
Molecular studies using RAPD and ISSR were carried out for the
assessment of genetic stability and variability of in vivo mother
plant and in vitro regenerated plantlets of Aloe vera L.
The leaves of conventionally grown Aloe vera plants were collected
from nursery of H.E.J Research Institute of Chemistry and
leaves of in vitro propagated plants were taken from the growth
room of Plant Tissue Culture and Biotechnology Wing.
The Aloe vera plant DNA was extracted using (CTAB)
method. Plant material was grinded using liquid nitrogen. Liquid
nitrogen is frequently used in DNA extraction protocols as it facilitates
grinding of the tissue by turning it into solid form and
has an additional advantage of maintaining low temperature. Many
small laboratories of developing countries faced the problem of
unavailability of liquid nitrogen. Storage and maintenance of liquid
nitrogen is also difficult. The highly versatile Cetyl trimethyl
ammonium bromide (CTAB) method (Sambrok, et al., 1989) is
a standard method for the extraction of DNA from various plant
materials. The availability of high-quality genomic DNA is a crucial
prerequisite for molecular genetic analysis of crops. There are
three main contaminants associated with plant DNA that can cause
considerable difficulties when conducting PCR experiments: polyphenolic
compounds, polysaccharides and RNA. Extraction of intact,
high molecular-weight DNA that can support PCR, genomic
blot analysis, fingerprinting and other molecular analysis is a challenge
when the plant tissue is rich in polysaccharides, secondary
metabolites or polyphenolics.
It is necessary to isolate good quality DNA that is relatively
free from many contaminants found in plant cells. Many
plant species contain characteristically high amounts of proteins,
polysaccharides, polyphenols (Angeles et al., 2005) and other secondary
metabolites, substance known for binding firmly to nucleic
acids during DNA extraction and interfering with subsequent reactions
(Ribeiro and Lovato, 2007).
Total cellular/genomic DNA isolation was performed
by classical cetyltrimethyl ammonium bromide (CTAB) method
described by Doyle and Doyle (1987) with some modifications.
To obtain good quality DNA, the utilization of fresh and young
leaf tissue is ideal (Ribeiro and Lovato, 2007). Latex gloves were
worn constantly during the DNA extraction to minimize the risk of
contamination of samples with nucleases from the skin and to protect
the skin from hazardous chemicals such as phenol, chloroform
and liquid nitrogen. Goggles were worn during the procedure to
protect eyes especially during grinding. About 1.5 g of Aloe vera
leaf samples were cut into small pieces and ground to fine powder
in pre-chilled pestle and mortar. Fine powder was suspended/homogenized
into 2 mL preheated (65°C) CTAB buffer (2% CTAB,
1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0 and 2% mercaptoethanol), kept on hot plate at 600C for 40 minutes to homogenize
and poured into 15 mL falcon tubes. The homogenate
was incubated at 65°C for 1 hour in shaking water bath after then
2 ml of fresh chloroform: iso-amyl alcohol (24:1, v/v) was added
and gently mixed by inverting the tubes for 50 times until there
was no interface. After then mixture was centrifuged (Eppendorf®
Centrifuge 5804R) at 10,000 rpm for 10 min at 40
C to separate
The supernatant/aqueous phase was collected in other
falcon tubes and 1:1 ratio of ice cold isopropanol was added to
precipitate the DNA. Isopropanol was mixed by shaking the tube,
when precipitation was not obvious the samples were kept at -200 C for an hour in freezer. After 1 hour the DNA was spooled with the
help of wire loop. In case, if DNA was not spool able then it was
again centrifuged at 10,000 rpm for 23 min at 40
C. The DNA made pellet at the bottom of the tube. The spooled DNA was put into 5
mL washing buffer for 20 min, DNA was collected from washing
buffer, air dried and then resuspended in TE buffer into labeled
microfuge tubes. The DNA dissolved/eluted into 1 mL TE buffer,
vortexed and left for overnight. Upon extraction of total genomic
DNA of each cultivar was given an extraction code and transferred
to eppendroff tubes.
Next day DNA samples were vortexed, added 10 µl
RNase A and incubated at 370C for 20 min. After incubation the
samples were centrifuged (Biofuge pico Heraeus) at 13,000 rpm
for 10 min. The supernatant was extracted; finally DNA quantification
was detected on 0.8% agarose gel (Wang et al., 2012) and by
UV spectrophotometer. All the coded eppendorff tubes were kept
in the freezer at – 20,. at Molecular Biology and Quality Control
Laboratory, Plant Tissue Culture and Biotechnology Wing, International
Center for Chemical and Biological Sciences, University
of Karachi-Karachi-75270, Pakistan. The remaining leaf samples
were kept in -200C freezer.
The nucleic acid concentration was determined using a
spectrophotometer (Schimadzu, 2000). Silica (Quartz) Ultra Micro
Spectrophotometer cuvette was used for holding the samples
and T.E buffer (10 mM Tris Hcl (pH 7.5), 1 mM EDTA) solution
was used as a standard/blank for calibrations of the spectrophotometer
at 260 nm and 280 nm. The DNA concentrations were
measured in spectrophotometer using 1:1000 dilutions. Readings
were recorded for each DNA sample at wavelength of 260 nm and
280 nm. An O.D. of 1 corresponded to approximately 50µg ml-1
double stranded DNA. The ratio of the readings was noted at 260
nm and 280 nm provided an estimation of the purity of nucleic
acids. The readings at 260 nm were used to calculate the DNA
concentration in the samples, by using the formula:
DNA concentration (µg/ml) = A260 x 50 x dilution factor (100)
An optical density value 1.0 corresponds to approximately
50 µg/mL for double stranded DNA. Pure samples of DNA have
ratios of 1.8 to 2.0 at O.D. 260/O.D. 280 and values of less than
this for DNA samples of lower purity. If there is contamination
of protein or phenols the ratio will be significantly lower than 1.8
and higher ratio signify contamination by RNA (Sambrook et al.,
1989). Besides this conventional method of DNA estimation was
performed by gel on 0.8% with λ DNA. The amount of plant genomic
DNA was estimated by visual comparison of band intensity
between λ DNA and plant DNA under following ethidium bromide
staining. DNA samples were diluted in T.E buffer to a working
concentration of approximately 10 ng µl-1.
The sequences of the different primers RAPD (octamer
oligonucleotide) used in this study are given in Tables 10b.
Molecular size of PCR amplified products were estimated
by using 1kb (Invitrogen) and 100 bp (Fermentas) DNA ladder.
DNA Amplification by RAPD-PCR
PCR amplification was carried out in Hi-Temp 96 wells
Thermal cycler (Master Cycler, Eppendorf, Germany). The following
concentration of PCR reagents (Fermentas, USA) were used
for 25 µl final reaction volume (Table 1 a). The cycling parameters
consisted of 30 min denaturation at 940
C, followed by 45 cycles
of 30 sec at 940
C, 1 min at 320
C, and 2 min at 720
annealing and extension).
The reaction was finally incubated at 720
C for 4 min, followed by soaking period at 40C until recovery. The lid temperature was kept constant at 1090 C.
Agarose Gel Electrophoresis :
Gel agarose was prepared in order to verify the presence
and the size of PCR products (amplicons) and 1 Kb and 100 bp
markers were used to calculate the molecular weight of the amplified
PCR products. The procedure was performed as following:
Melting of the Agar
Molecular Biology grade agarose (Gene-Link and Scharlu)
was used at 1 % for the separation of amplified PCR products. The agarose was suspended in the 1x TBE (Tris Borate EDTA)
buffer pH 8.0 in a flask. The flask was covered with another small
mouth flask as the vapors can come back again in the same flask of agarose. The solution was heated in microwave oven (Sharp 1000W/R-2197), till the agarose dissolved completely and solution
become clear. During boiling the solution was gently swirled
to re-suspend any settled agarose. The flask was removed carefully
using thick hand gloves/protector. Then gels were allowed to cool at about 600C, as the flask can be handled comfortably. 5 µl
ethidium bromide (0.05 µg/mL) was mixed properly in the agarose
Pouring the Gel
The electrophoresis gel tray/plate was prepared and a
comb was placed in it to make loading wells. The cooled agarose
gel was poured/casted into gel tray containing a comb. The agarose
should come at least half way up the comb teeth. The agarose gels
were allowed to solidify for 40 min after than comb was removed.
Setting up the Gel Tank
After solidification gel was placed in the appropriate
electrophoresis tank, the wells were placed to negative electrode
and covered with 1X TBE running buffer. The DNA samples/
PCR products were mixed with gel loading dye (0.25% (w/v)
Bromophenol Blue, 40% (w/v) sucrose, 0.1mM tris, and 0.05 mM
EDTA) in a 1:4 ratio. The bromophenol blue dye give density to
PCR product as the sample can sink to the bottom of the well properly
and PCR product 8 µl plus 2 µl of loading dye mixed properly
and loaded into each well created by comb. One (1) Kb ladder
(Life TechnologiesTM) used as molecular size marker or gene ruler
on each gel.
Running and Analyzing the Gel:
The lid was placed on the tank and the electrode leads
were connected to the power supply. The electrophoresis was generally
conducted by using large submarine units (Thermo EC-320)
at 60V for 2hr (Thermo EC, EC-250-90). Then gels were removed
from the electrophoresis tank and placed in gel documentation system
(UV TechTM, UK), images of DNA bands obtained and photographed.
The photos were saved in specific file till to be used.
The PCR reactions were repeated twice for the confirmation of
Results And Discussion
Evaluation of Genetic Stability of Aloe vera plants by
RAPD Marker Assay Among multilocus markers RAPD and ISSR
techniques have been widely used to detect, evaluate and identify
changes in the DNA sequence caused by somaclonal variation in
Aloe (Alagukannan & Ganesh, 2016; Kumar et al., 2016). Molecular
characterization using PCR based technique Random Amplified
polymorphic DNA (RAPD) was conducted on conventionally
grown (wild type) and tissue cultured Aloe vera plants to compare
their genetic similarity/diversity. Eight primers were selected for
the analysis as they were reported to produce polymorphic amplicons
as shown in Figure 1 and Table 2a.
The primer LC-76 generated total 2 monomorphic bands
with DNA templates of both conventional and tissue cultured
plants and showed 0% polymorphism. Both Primers LC-77 and
LC-83 produced total 4 bands with 3 monomorphic and 1 polymorphic
band, each exhibiting 25% polymorphism. Primer LC-
81 produced all 4 monomorphic bands and no polymorphism was
seen. LC-87 produced total 5 band with 3 monomorphic and 2
polymorphic amplicons, showing 40% polymorphism. While LC-
90 produced total 5 amplicons with 4 monomorphic and 1 polymorphic
bands giving 20% polymorphism. OPL-1 generated 5
monomorphic bands with no polymorphism. OPQ-12 produced
highest number of bands (9), of which 6 were monomorphic and
3 polymorphic exhibiting 66.6% polymorphism. Thus with both
types of Aloe vera DNA templates, all the eight RAPD primers
generated 4.75 amplicons on the average, of which 3.75 were
monomorphic and 1 polymorphic, showing 22% polymorphism.
The DNA bands produced by individual wild type and in
vitro cultured plants with all the eight bands was also calculated
(Table 2a). With all the eight primers tested, conventionally grown
Aloe vera plants produced total 29 band, showing minimum 1 and
maximum 6 bands. However DNA templates of tissue cultured
plants generated 32 bands with all the primers giving minimum 1
and maximum 9 bands.
Rathore and his coworkers in 2011, published the first report
on the comparison of genetic stability/instability of tissue-culture
Aloe vera plants exploring two regeneration systems. No
polymorphism was observed in regenerates produced from direct
regeneration system from axillary buds, whereas 80% polymorphism
was observed in plants produced through indirect regeneration
via intermediate callus phase. Our results are in harmony with
the previous findings (Das et al., 2016), as our Aloe vera plant regenerants
produced through indirect regeneration via callus phase,
have exhibited polymorphism in a range of 20-33% (Table 2b).
Figure 1: RAPD profiles of Aloe vera wild type soil grown and tissue cultured plants with primers LC-76, LC-77, LC-81, LC-83, LC-
87, LC-90, OPL-1 and OPQ-12.
Table 2a: Total number of bands calculated from tissue cultured and wild type Aloe vera plant with eight RAPD primers
Table 2b:Polymorphism percentage between tissue cultured and soil grown Aloe vera plant by using 8 RAPD primers
Evaluation of Genetic Stability of Aloe vera plants by
ISSR Marker Assay
During the last decade new development in the PCR based techniques
on DNA markers are used as powerful tools to validate the
genetic fidelity of in vitro plants (Butiuc-Keul et al., 2016; Shahzad
et al., 2017). The present study was aimed at analyzing the genetic
similarity and diversity in wild type field gown and tissue cultured
Aloe vera plants by using ISSR markers. ISSR is a type of molecular
markers based on inter-tandem repeats of short DNA sequences.
These inter repeats are highly polymorphic, even among
closely related genotypes, due to the lack of functional constraints in these nonfunctioning regions. ISSR markers are simple, more
reliable and proved highly efficient in analysis of genetic diversity
studies (Nookaraju and Agrawal, 2012; Das et al., 2016). The analysis
was carried out with 4 inter simple sequence repeat (ISSR)
markers, which were screened out for their effectiveness and reproducibility.
The primers successfully produced clear bands with
all the DNA templates of Aloe vera (Table 3a and Figure 2) as
Figure 2: ISSR profiles of Aloe vera wild type soil grown and tissue cultured plants with primers ISSR-A, ISSR-G, ISSR-M and ISSR-O.
Table 3a: Total number of bands calculated from Tissue culture and Wild type Aloe vera plant with four ISSR primers
Tissue cultured plants produced total 18 bands with all the primers and 17 bands of wild type plants were produced. Of all the primers
tested only primer ISSR-G exhibited 25% polymorphism with 3 monomorphic and 1 polymorphic band. The other primers did not produce
any polymorphic band. On the average 4.25 monomorphic band were generated with all the four markers with a range of minimum
3 and maximum 6 amplicons (Table 3b).
Combined Analysis of RAPD and ISSR Markers
In the present study polymorphism level of conventionally grown
wild type and in vitro propagated Aloe vera plants was investigated
using RAPD and ISSR based molecular markers (Table 4).
Tissue cultured plants produced maximum number of bands (32)
with all RAPD primers and 18 bands with ISSR primers. Wild
type field grown Aloe vera plants produced 29 DNA bands with
all primers of RAPD and 17 bands with ISSR primers. Thus total
50 DNA bands of tissue culture plants were generated from all the
12 primers of both molecular markers, whereas total 46 bands of
conventionally grown plants were generated. In sum up of all the
two molecular markers with both Aloe vera plants total 94 bands
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