(RAPD) markers

In vitro variability of Cymbidium sp. based on random
amplified polymorphic DNA (RAPD) markers
Petolescu Cerasela 1 *, Lazar A. 1
1 USAMVB Timisoara, Faculty of Horticulture and Sylviculture
*Corresponding author. Email: cerapetolescu@yahoo.com
Abstract The objective of this study was to determine in vitro variability
using randomly amplified polymorphic DNA (RAPD) markers. DNA from
eleven in vitro obtained plantlets of cymbidium was examined using
polymerase chain reaction (PCR) to determine the efficiency of randomly
amplified polymorphic DNA (RAPD) markers in identifying of somaclonal
variability. We used 4 RAPD primers. Concerning RAPD analysis, template
DNA produced clear PCR profiles. As a result of UV light screening of
agarose gels, it has been observed slight differences between the genetic
fingerprints of the studied variety induced by amplification using RAPD primer.
Key words
Cymbidium, RAPD,
somaclonal variation
Cymbidium is one of the Orchidaceae genera.
There are 70 Cymbidium species in the world, mainly
distributed over the tropical and the subtropical area of
Asia and northern Australia. The genus Cymbidium
(Orchidaceae) comprise species distributed from
Northwest India to China and Japan, south through the
Malay Archipelago to North and East Australia.
Cymbidiums have been, for over one hundred years,
and remain at the present day the most important
orchid in commerce, being primarily grown for their
flowers which are attractive, long lasting and large.
They are increasingly gaining popularity as decorative
pot plants with smaller, but equally high quality
flowers. Most of the commercially important, large
flowered cymbidiums in cultivation are hybrids derived
from the 44 recognised species. These modern hybrids
are often complex, involving several species in their
ancestry.
Classical approaches for the identification of
cymbidium cultivars are based on morphological traits.
The assessment of these traits is difficult and their
evaluation can be subjective considering that most of
these cultivars are related.
Many cultivars have been selected and used;
however, their genetic background was unknown.
Genetic diversity has been studied in plant species
using a variety of morphological, chemical and
molecular descriptors.
The random amplified polymorphic DNA
technique (Welsh J and McClelland M (1990),
Williams et al., 1990) based on the polymerase chain
reaction (PCR) has been used for in vitro variability
identification and or genetic diversity studies in roses
peach, tea, cymbidium [9,10,11] and annanas. The
RAPD technique has also been used to determine the
purity of hybrid seed in tomato.
Molecular markers and molecular sequences
contain useful information about evolutionary
history.[8,12].Recently, the use of random amplified
polymorphic DNAs (RAPDs) has become popular. The
arbitrarily primed polymerase chain reaction (or
RAPD) amplifies anonymous fragments of DNA from
any genome [2,3]. The size distribution of amplified
fragments varies among species. However, closely
related species have similar fragment distribution,
while distantly related ones are more divergent[4]
Thus, RAPD bands (fragments) distribution contain
considerable phylogenetic information[5,6].
Biological Material and Method
The biological material used in this study was
represented by cymbidium plantlets obtained in vitro
culture. Culture media that have generated best results
was a variant of Murashige-Skoog media[1] added
with an auxin, α-naphthalene acetic acid (2 mg/l) and a
cytokinin, 6-benzylaminopurine (1,5mg/l). Resulting
plantlets were tested for genetic variability and
somaclonal variation using RAPD analysis.
We used four RAPD primers (G04, G10, G17, G18)
with following sequences: 5’AGCGTGTCTT 3’ 5’
AGGGCCGTCT 3’, 5’ ACGACCGACA 3',
5’GGCTCATGTG 3’. Total genomic DNA was
extracted from each plantlets leaf tissues using a
modified CTAB method. DNA samples were diluted in
TE buffer and submitted to electrophoresis (3V cm-1)
in 0.7% agarose gels (w/v). DNA was stained by gel
immersion into ethidium bromide solution for 30 min.
RAPD reactions were performed in a final volume of
25 µl in PCR buffer containing MgCl2, RAPD primers,
dNTP, DNA template and Taq DNA polymerase.
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Reactions were submitted to the following PCR
program: preliminary DNA denaturation for 3 min at
94°C, followed by 45 cycles consisting of denaturation
(3 min, 94°C), primer annealing (1,5 min, 36°C), and
extension (2 min, 72°C). A final extension for 2 min at
72°C was included. The RAPD products were
separated by electrophoresis (3V cm-1) in 2% agarose
gels, which run with 1 x TAE buffer. Photo
documentation was performed under UV light using a
photo imaging system.
Results and Discussions
This research wants to show, if in this case, it
induced somaclonal variation.The frequency of the
RAPD fragments was estimated for each individual.
The total number of clear bands obtained from each
primer ranged from 2 (G03) to 7 (G10).
Concerning RAPD analysis, template DNA
produced clear PCR profiles. As a result of UV light
screening of agarose gel, it has not been observed
differences between the genetic fingerprints of the
studied variety induced by amplification using GO4,
G17, G18 primers. The amplification using G10
primers show some differences, emphasizing slight
polymorphism at molecular level. We can see a gel
with a size marker on the left (M) and 12 individuals
analyzed with G10 primer. The size of bands varied
from 1000 bp-300 bp. It can be observed a slight
polymorphism at molecular level. Six individuals
present differences, it identify the presence a band by
400 bp. It can be observed that are differences
comparing with the mother-plant.
M mt 1 2 3 4 5 6 7 8 9 10 11
Figure 1. RAPD patterns generated by primer G10
Legend: M – PCR marker ( 700, 500, 400, 300, 200, 150, 100, 75, 50, 25 bp);
mt - mother plant; lanes 1- 11in vitro regenerated plants
M mt 1 2 3 4 5 6 7 8 9 10 11
Figure 2. RAPD patterns generated by primer G18
Legend: M – PCR marker ( 700, 500, 400, 300, 200, 150, 100, 75, 50, 25 bp);
mt - mother plant; lanes 1- 11in vitro regenerated plants
We can see a gel with a size marker on the left
(M) and 12 individuals analyzed with G18 primer. The
size of bands varied from 500 bp-195 bp. It it has not
been observed polymorphism at molecular level.
It can be observed that are not differences
comparing with the mother-plant.
We can see a gel with a size marker on the left
(M) and 12 individuals analyzed with G17 primer. The
size of bands varied from 550 bp-500 bp. It has not
been observed polymorphism at molecular level. It can
be observed that are not differences comparing with the
mother-plant.
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M mt 1 2 3 4 5 6 7 8 9 10 11
Figure 3. RAPD patterns generated by primer G17
Legend: M – PCR marker ( 700, 500, 400, 300, 200, 150, 100, 75, 50, 25 bp);
mt - mother plant; lanes 1- 11in vitro regenerated plants
Conclusions
Data presented and discussed in this study
represent a first step for the next studies concerning the
efficiency of randomly amplified polymorphic DNA
(RAPD) markers in identifying of somaclonal
variability of cymbidium. In the future study we will
study more plants and we use more RAPD primers. Ex
vitro Cymbidium plantlet genetic stability will be also
tested and compared.
The evaluation of somaclonal variability of
cymbidium using RAPD markers alloed to draw the
following conclusions:
- It has not been observed differences between
the genetic fingerprints of the studied variety
induced by amplification using GO4, G17,
G18 primers. It can be observed that are not
differences comparing with the mother-plant
- The amplification using G10 primers show
some differences, emphasizing slight
polymorphism at molecular level.
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