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DNA Fingerprinting of S
ix
S
enna

Species and the Taxonomic I
mplication.

J
imoh
, M. A., E
deoga
, H. O. AND *O
mosun
, G.

Department of Plant Science and Biotechnology, Michael Okpara University of
Agriculture, Umudike, Abia State, Nigeria.

ABSTRACT

DNA fingerprinting of six medicinally important
Senna

species was done so as to aid

easy recognition of the
Senna

species for pharmacognostic researches. This study was
carried out in order to eliminate adulteration of these medicinally important
Senna

species by
misidentification.
The species studied were
S. alata, S. obtusifolia, S.
siamea, S. hirsuta, S. occidentalis
and

S. polyphylla.

In this study, random amplified
polymorphic DNA (RAPD) was used to develop markers for authentication of these
Senna

species.
From the RAPD analysis, a dendogram was generated from the
detection of polymorphic fragments in the six primer sequences that was analysed, the
dendrogram showed that all the species are at least 62% similar.
S. alata

and
S hirsuta

were 95% gene
tically identical and far from other accessions in terms of similarity.
This is common in speciation. The RAPD analysis led to a clear distinction between
S.
alata

and
S. hirsuta

and other species
.

The present study has shown that RAPD marker
technique is
very useful for genetic variability and species relationship in the genus,
Senna
. One reason is that this method is typically independent of environmental
interactions in contrast to morphology, anatomy and phytochemical methods of
identification.
RAPD cou
ld thus, serve as a complementary tool for quality control of
the
six
Senna

species

*Corresponding Author

Email:
omosun.garuba@mouau.edu.ng

Phone: +2348063806575




INTRODUCTION

The genus
Senna

comprises shrubs,
sub shrubs
, and herbaceous perennials with
paired
-
pinnate leaves.
Senna

is native to tropical Africa and cultivated in Egypt and the
Sudan and elsewhere; it is native to India and cultivated mainly in India and Pakistan
(Boulos, 2002).
S
enna

is a stimulant laxative and used for treatment of constipation
(Bradley, 1992). Significant inhibitory activity in mice against leukemia has been
documented.


In Nigeria, the

plants are used for treating eczema and other skin

defects caused
by fungal
infections (Akinremi
et al
., 2000), the leaves are also prepared into vegetable

soup for small pox and measles.
Senna podocarpa
leaves are extensively known for
their anti
-
gonorrhoeal

and purgative properties as well as a guinea worm and sore
-
healing remedy among the Igbos in Nigeria (Akanmu, 1999). Fresh leaves concoction
are used for curing syphilis (Gomes
et al
., 1997), herpes and swine fever (Silva
et al
.,
1997) used as purgative

and for repelling or killing insects such as termites, bed bugs
and mosquitoes (Elujoba
et al
., 1999).

One of the most reliable methods for identification of medicinal plants involves
morphological and genetic analysis. Molecular techniques have been
also introduced
for DNA fingerprinting (Sucher and Charles, 2008). Analysis of the DNA that is
present in all organisms is a suitable method for identifying plant materials because the
genetic composition is unique for each individual organism. DNA extract
ed from
leaves, stems or roots of plants all carry the same genetic information without being
affected by physiological conditions and environmental factors. Polymerase Chain
Reaction (PCR) in combination with Restriction Fragment Length Polymorphism
(PCR/
RFLP) has been widely used for DNA fingerprinting (Weis
l
ing
et al
, 2005).
Species
-
specific regions in nuclear DNA, mitochondrial DNA and chloroplast DNA
have been used for identification of individual species. The Internal Transcribed Spacer
(ITS) region,
found on either side of the transcribed region of the 18S, 5.8S and 26S
nuclear ribosomal DNA, is one of the most popular sequences for phylogenetic analysis
of plants for species
-
level identification (Sukrong
et al
., 2007). The ITS region is
highly repeat
ed in the plant nuclear genome which is present in the form of up to many
thousands of copies arranged in tandem repeats.

DNA fingerprinting is an important tool for molecular characterization of various
groups of plants. It offers a faster and more preci
se way of determining relationships
among closely related species than that of morphological investigation (Rahman, 2007),
this is because morphological characteristics are subject to environmental influence and
extensive studies of mature plants are often

necessary for taxonomic classification. The
molecular assay is undertaken to evaluate the potential of PCR based fingerprinting for
detecting genetic variation and inferring molecular classification among some species
of
Senna
.

On this background, the pre
sent study
is the DNA analysis using PCR method

for
fingerprinting of

six
Senna

species

(family Leguminosae: Caesalpiniodeae) in Nigeria
.


Materials and Methods

PCR (Polymerase Chain Reaction) for RAP
D (Random Amplified Polymorphic
DNA
) analyses

DNA Extraction from
Senna

plant leaf.

DNA was isolated from the leaves using the CTAB (Cetyltrimethylammonium
bromide) method described by Doyle and Doyle (1987). About 0.5 g of leaf sample was
put into eppendorf tube, then 500µl CTAB buffer was added. The

plant tissue was
homogenized in the buffer by grinding. 75µl of 10% SDS was added. The sample was
then incubated at 65
0
C for 30 minutes. It was allowed to cool. After cooling, 10µl
Proteinease K was added and then incubated at 37
0
C for 30 minutes. 500µl C
hloroform
was then added and mixed for about 5 minutes. It was spinned at 10,000rpm for 10
minutes. The supernatant was collected into

fresh eppendorf tube without taking the
white interphase.

To the supernatant, 500µl volume of 100% ethanol was added, k
ept at
-
20
0
C for 1
hour. It was then spinned at 10,000 rpm for 10 mins, the supernatant was decanted
gently and 500µl 70% ethanol was added and smixed thoroughly. The mixture was
spinned at 10,000 rpm for 10 minutes and the supernatant discarded. The pelle
ts
obtained were air dried for 30 minutes. The pellets were then re
-
suspended in 200µl
sterile water.

Dilution of DNA for PCR

RAPD reaction was performed according to the method developed by McClelland
et al.,

(1995). About 10µl of each extracted DNA was taken into eppendorf tube with
990 µl sterile distilled water to have 20


50ng/
µl (1:100 dilution).

PCR Mix (RAPD PRIMERS)


10X Buffer


2.0ul

25mMMgCl
2

1.6ul

5%Tween20


2.0ul

2.5mMdNTPs

1.0ul

2.0mMPrimer

1.0ul

5u/ulTaq


0.2ul

Water


8.2ul

Diluted DNA

4.0ul


PCR Profile

3mins


94
o
C


1cycle

20sec


94
o
C}

20sec


37
o
C}


45 cycles

40sec


72
o
C}

7mins


72
o
C


1 cycle

Gel electrophoresis

1.4% agarose

was prepared using 2.8g in 200mls 1X TAE, microwave to dissolve
the agarose and allow to cool to room temp. Before it was poured into the gel tray and
allowed to solidify. The samples and markers were loaded. The sample was run for
2hours at 150V and 0.5
miniamp.

Staining

The gel was stained in 2.5mg/L Ethidium Bromide for 3minutes and further
distained for 10 minutes in sterile water and then viewed under UV light and the picture
was taken.

Data Analysis

Each band was considered as a character and for each species, the presence or
absence of band was scored in a binary code (present = 1, absent = 0). A data matrix
was generated using the binary code. Pair
-
wise similarity coefficients were generated
by usin
g the Simqual subprogram of NTSYS
-
pc Version 2.1

(Rohlf, 2000) and used for
cluster analysis with the SHANsubprogram of NTSYS
-
pc. A dendrogram was created
based on the Unweighted Pair GroupMean

Average (UPGMA) method. For the genetic
distance coefficient calculation, the Nei and Li
(1979)
Matching Coefficient w
as

used.

Results and Discussion

The polymorphism obtained in these six
Senna

species showed a distinct
variation. The highest degree of
similarity was between
S. alata
and

S. hirsuta

(0.95),
while the lowest (0.58) was observed between
S. occidentalis
and

S. polyphylla
(Table
2).

The dendogram (Fig. 1) was generated from the detection of polymorphic
fragments (allelic variants seen in th
e bands) in the six primer sequences that was
analysed. From the dendrogram all the accessions are 62% similar.
S. alata
and

S
hirsuta

are 95% genetically identical and far from other accessions in terms of
similarity. This is common in speciation. The RAP
D analysis led to a clear distinction
between
S. alata and S. hirsuta
and other species. Tripathi
et al.,

(2009) similarly used
RAPD analysis to separate
S. tora
and

S. obtusifolia
. The RAPD analysis has been
widely used for differentiation of a large numb
er of medicinal species from their close
relatives (Khan
et al
., 2011
). The advantage of RAPD techniques is their simplicity,
rapidity, requirement of low amount of genomic DNA and avoidance of radioactive
substances (Micheli
et al
., 1994).

DNA fingerprin
ting employing different marker systems is quite evident these
days (Roy
et al.,

2006). The RAPD marker may prove to be reproducible under a wide
variation of the amplification conditions such as an annealing temperature, origin of the
primer,
Taq

polymera
se and thermal cycler. In this study, The RAPD technique used
for the fingerprinting and identification of the six
Senna

species is convenient since the
study showed a clear discrimination between S.
alata

and S.
hirsuta

from the other four
Senna

species.







1



2




3


4




5


6


M




Plate 1
: RAPD analysis carried out with primer OPAD
-
09 on genomic DNA
extracted from the leaf of the six
Senna

spp. DNA Fragments size range from
200bp

-

1500bp.





M




1




2


3




4


5


6




Plate
2: RAPD analysis carried out with primer OPAE
-
04 on genomic DNA
extracted from the leaf of the six
Senna

spp. DNA Fragments size range from
250bp
-

2500bp






1

2


3


4


5


6



M




Plate
3: RAPD analysis carried out with primer OPAE
-
05 on genomic DNA
extracted from the leaf of the six
Senna

sp
p. DNA Fragments size range from
150bp
-

2000bp


.



1


2


3


4


5


6




M




Plate
4: RAPD analysis carried out with primer OPAF
-
07 on genomic DNA
extracted from the leaf of the six
Senna

spp. DNA Fragments size range from
250bp
-

3000bp






1


2




3

4



5


6

M





Plate
5: RAPD analysis carried out with primer OPAF
-
09 on genomic DNA
extracted from the leaf of the six
Senna

spp.

DNA Fragments size range from
200bp
-

1500bp.


Table

1: Primers used and DNA fragment size.


S/N

Primer Name



Sequence



Size range

1

OPAD
-
09


TCGCTTCTCC

200bp
-

1500bp

2

OPAE
-
04


CCAGCACTTC

250bp
-

2500bp

3

OPAE
-
05


CCTGTCAGTG

150bp
-

2000bp

4

OPAF
-
07


GGAAAGCGTC

250bp
-

3000bp

5

OPAF
-
09


CCCCTCAGAA

200bp
-

5500bp






Table
2

Similarity Coefficients of the six species of
Senna


S. alata

S. hirsuta

S. polyphylla

S. siamea

S. occidentalis

S. obstufolia

S.alata

1.0000000






S.hirsuta

0.9454545

1.0000000





S.polyphylla

0.6909091

0.6363636

1.0000000




S.siamea

0.8000000

0.7818182

0.6727273

1.0000000



S.occidentalis

0.7818182

0.7636364

0.5818182

0.6909091

1.0000000


S.obstufolia

0.8545455

0.8363636

0.7272727

0.8000000

0.7818182

1.0000000




Fig.
1:
Dendogram of genetic similarity for the six
Senna

species


Many authors have worked on the ethnobotany, phytochemical, antimicrobial and
morphological aspect of the
Senna

species (Ogunkunle and Ladejobi, 2009, Ogundipe
et al
., 2009, In
gwenye
et al
., 2010,
Ogundare, 2009). However very little information is
available on the molecular studies of this genus. Although a previous study
demonstrated the genetic variation between
S. tora
and

S. obtusifolia

(Tripathi
et al
.,
2009). The present study has shown that
RAPD marker technique is very useful for
genetic variability and species relationship in the genus,
Senna
. One reason is that this
method is typically independent of environmental interactions like morphology,
anatomy and phytochemicals studied. The presen
t study has shown that RAPD marker
technique used have shown that the
Senna

species investigated are closely related.

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