exploration of genotype specific fingerprinting of nigella sativa l. using rapd markers
TRANSCRIPT
M. S. IQBAL, S. NADEEM, S. MEHBOOB, A. GHAFOOR, M. I. RAJOKA, A. S. QURESHI, B. NIAZ
569
Turk J Agric For
35 (2011) 569-578
© TÜBİTAK
doi:10.3906/tar-1001-622
Exploration of genotype specifi c fi ngerprinting of
Nigella sativa L. using RAPD markers
Muhammad Sajjad IQBAL1,2,5,
*, Shahid NADEEM2, Shahid MEHBOOB
2, Abdul GHAFOOR
3,
Muhammad Ibrahim RAJOKA2, Afsari Sharif QURESHI
1, Bushra NIAZ
4
1Genetics Laboratory, Department of Biochemistry, Quaid-I-Azam University, Islamabad, 46000 - PAKISTAN
2Department of Bioinformatics, GC University, Allama Iqbal Road, 38000, Faisalabad - PAKISTAN
3Institute of Agri-Biotechnology & Genetic Resources, National Agricultural Research Center,
Islamabad, 45500 - PAKISTAN4Department of Post-Harvest, Ayub Agriculture Research Institute, Faisalabad - PAKISTAN
5Department of Genetics, Hazara University, Mansehra, 21300 - PAKISTAN
Received: 23.01.2010
Abstract: Nigella sativa L. has industrial, cosmetic, and pharmaceutical uses but has not been adequately characterized
in Pakistan. Th is investigation was carried out to explore genotype specifi c fi ngerprinting of 32 N. sativa L. genotypes
based on randomly amplifi ed polymorphic DNA markers. From 58 random primers used, 15 primers generated 249
reproducible and scorable amplifi cation products across all the genotypes, out of which 164 (66%) fragments were
polymorphic revealing a high level of polymorphism among these genotypes. Th e proportion of common bands was low
(34%). Th e size of the amplifi cation products on agarose gels ranged between 0.5 and 10.0 kb. In 13 genotypes, 27 bands
of diff erent masses (kilobases) were recorded and were considered specifi c to those genotypes. Th ese specifi c/amplifi ed
PCR products can be used as molecular markers for identifi cation of germplasm and resource protection of Nigella
sativa L. genotypes. Specifi c bands were observed for individual primers that could resolve genetic diversity among
several genotypes (PK-020545, PK-020567, PK-020576, PK-020585, PK-020592, PK-020620, PK-020631, PK-020646,
PK-020663, PK-020729, PK-020742, PK-020749, and PK-020868). UPGMA cluster analysis indicated 7 distinct clusters,
1 (C-3) comprising 9 accessions of N. sativa L., while C-7, C-5, and C-6 included 7, 6, and 5 accessions, respectively. C-4
and C-2 included 2 accessions each. Cluster 1 remained distinct as it had only 1 accession (PK-020646), indicating its
higher genetic diversity from all other species. Th e overall grouping pattern of clusters corresponded well with principal
component analysis and confi rmed overall patterns of genetic variability among the species. Th ese genotypes could
be used as parents for random mutagenesis, or incorporated for gene recombination studies before marker assisted
selection/breeding can be used for crop improvement. Moreover, these DNA based markers can be suitable for genetic
distance estimation because they detected potentially large number of polymorphisms. Future research is needed to
identify molecular markers linked to important traits (yield and oil quality) and locating quantitative traits loci (QTL)
for improvement of N. sativa L. germplasm.
Key words: Biodiversity, genetic relatedness, genotype specifi c bands, PCR, polymorphism, RAPD, random priming,
UPGMA
Research Article
* E-mail: [email protected]
Exploration of genotype specifi c fi ngerprinting of Nigella sativa L. using RAPD markers
570
Introduction
Th e Ranunculaceae or buttercup of the order Ranales is a large family containing about 70 genera and at least 3000 species. Th e genus Nigella contains about 20 species of annual herbs indigenous to the Mediterranean region through West Asia to Northern India, and has long been domesticated (Weiss 2002). Nigella sativa L. (diploid, 2n = 12) is an erect annual herb considered important for both oil and bioactive compounds because its constituents have unique chemical properties and may augment the supply of edible oils or production of biodiesel (Ramadan and Morsel 2003; Iqbal et al. 2010).
Genetic variability is a prerequisite for selection program of germplasm; it is essential to detect and present the level of genetic variation present within and between plant populations. DNA marker-assisted fi ngerprinting can diff erentiate species rapidly employing a small quantity of DNA and therefore can help to derive reliable fi ndings on their phylogenetic relativity. DNA markers are not infl uenced by environmental impacts and are useful in describing the levels of genetic variability among plant populations to discriminate the duplicated accessions within a specifi c collection of germplasms. Many researchers have used diff erent techniques for DNA fi ngerprinting such as restriction of length polymorphism (RFLP), amplifi ed fragment length polymorphism (AFLP), sequence characterized amplifi ed region (SCAR), simple sequence repeat (SSR) markers, and randomly amplifi ed polymorphic DNA (RAPD) markers in combination with the analysis of morpho-physiological traits in various crops (Rafalski et al. 1991; Williams et al. 1993; Cheng et al. 2002). RAPD is a rapid and inexpensive method and non-dependent on plant genome information. It has been extensively applied to ascertain genetic polymorphism in several plants (Murai and Ohnishi 1996; Nevo et al. 1998). Th e variability of RAPD markers was measured by the proportion of polymorphic bands, in the sense that some natural populations produce a particular band, but others do not. Th is measurement of variability is highly dependent on the number of samples studied and on the ecological origins from where the samples were collected.
RAPD involves the employment of short (10 bp) PCR primers of arbitrary sequence to amplify
unknown genomic sequences (Khan et al. 2002).
When the resulting products are separated in suitable
electrophoretic gels, DNA-level polymorphisms
(usually in primer recognition sites) can be uncovered
(Williams et al. 1990; Welsh et al. 1991). RAPD
techniques are still employed by many laboratories,
as easy ways of screening potential molecular
markers from many loci using small amounts of
DNA (Ritland and Ritland 2000). Moreover, DNA
fi ngerprinting is a powerful technique for assessing
genetic diversity levels among germplasms (Lee
1995). Crops with improved output traits could have
nutritional benefi ts for millions of people who suff er
from malnutrition or other defi ciency disorders.
Identifi cation has been carried out of all those genes
that can modify and enhance the composition of
carbohydrates (including starch), oils, and proteins
in food/feed grains and root crops. Th e development
of molecular markers based on polymorphism
observable in protein or DNA has greatly facilitated
research in phylogeny, ecology, plant breeding, and
genetics.
Information regarding DNA fi ngerprinting and
polymorphism in Nigella sativa L. germplasm is not
widely available and demands its exploration. Due
to applications of molecular markers, a number of
genomes were sequenced and studied for diff erent
purposes. Th ey were especially employed in the
fi eld of plant genetics and breeding, biotechnology,
and bioinformatics. Study of DNA fi ngerprinting
of Nigella sativa L. has been undertaken to explore
the genetic relationship between its diff erent species
using RAPD markers in order to exploit the present
germplasm effi ciently.
Materials and methods
Genomic DNA isolation
For good quality and purity, genomic DNA was
isolated/extracted following the method of Kang
et al. (1998) using seeds of all the 32 accessions.
Known weights of seeds (0.03 g each) were placed
in 1.5 mL microcentrifuge tubes (Eppendorf tubes).
Extraction buff er 400 μL (200 mM Tris-HCl, pH
8.0, 25 mM EDTA, 200 mM NaCl, 0.5% SDS) was
added to all samples. Proteinase K (50 μg) was
added to each tube to remove traces of proteins in
M. S. IQBAL, S. NADEEM, S. MEHBOOB, A. GHAFOOR, M. I. RAJOKA, A. S. QURESHI, B. NIAZ
571
order to get pure genomic DNA. Th en samples were incubated at 37 °C for 1 h. Th en seed samples were ground with the addition of buff er with a glass rod. Four hundred microliters of 2% CTAB buff er (100 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 2% CTAB (w/v), 1% PVP (polyvinyl pyrrolidone” Mr. 40,000) was added. Impurities usually create a problem during extraction of good quality DNA, especially in the case of medicinal plants, which have complex chemical compositions (Cheng et al. 2003). Th en 600 μL of chloroform:isoamyl alcohol (24:1) with 5% phenol was added and mixed gently (manually) for about 2 min to get a homogenous mixture. It was then centrifuged at 12,000 rpm at 4 °C for 10 min. Th e aqueous layer was now recovered carefully and shift ed into new 1.5 mL Eppendorf tubes. Seven hundred microliters of isopropanol, i.e. 2/3 (v), was added and incubated at room temperature for 10 min to precipitate DNA. Genomic DNA appeared in the form of threads. Th en Eppendorf tubes were centrifuged at 12,000 rpm for 10 min and this time supernatant was removed. Th e DNA pellet was washed with 70% ethanol (500 μL) and again centrifuged at 12,000 rpm for 5 min at room temperature to remove ethanol. Th e DNA pellet was air dried for 5-10 min and resuspended in 100 μL of TE buff er for storage, quantifi cation, and PCR application. RNase (10 mg mL-1) 1 μL was added to remove RNA.
Th e purity and concentration of extracted genomic DNA were checked by spectrophotometer (Model Agilent 8453, USA). DNA concentrations were calculated using the formula [DNA = optical density (OD
260) × dilution factor × constant
(50 μg mL-1)]. DNA samples were diluted to a working concentration of 50-100 ng μL-1 in sterile distilled water and stored at 4 °C. Th e integrity and concentration of the DNA was confi rmed by running 1% (w/v) agarose gel electrophoresis for 45 min at 80 V and visualization under UV light aft er staining with ethidium bromide.
PCR amplifi cation kit and RAPD analysis
For PCR-RAPD analysis, 10 to 12 base oligonucleotide primers were used, which were obtained from Operon Technologies Inc. USA (Table 1). PCR was performed by volume of 20 μL of 10× buff er, called master mix including 2 μL {[Tris-HCl (100 mM), pH 8.8 with (NH
4)
2 SO
4 (1 mL)], 1.6 μL
MgCl2 (25 mM, 1 mL), 0.4 μL dNTPs [each of dATP,
dCTP, dGTP, dTTP, (25 mM)], 0.8 μL of primer (15 ng μL-1), 1 μL of genomic DNA (1 ng μL-1), 0.15 unit of Taq polymerase and ddH
2O (13.85 μL)}. A total
of 58 primers were used for screening of RAPD markers. Taq polymerase, together with 10× buff er, MgCl
2, and dNTPs, were obtained from Fermentas
(Canada). PCR-based DNA amplifi cation reactions were performed in Tpersonal Biometra DNA Th ermocycler 480 (Germany).
Optimization of RAPD Protocol
Th e PCR-RAPD technology is sensitive to changes in experimental parameters. A total of 58 primers were initially screened against 10 plants selected from all populations (bulked seed DNA). Th e eff ects of magnesium, template DNA concentrations, pH values, and length of the denaturation stage of the amplifi cation were all examined. When trying to optimize annealing temperatures, we ran the test reactions at 27 °C, 30 °C, 33 °C, 36 °C, and 37 °C. Th e decamer primers can be clearly amplifi ed at 34 °C. A subset of 15 primers for further analysis was based on the following criteria: i) consistent, strong amplifi cation products, and ii) production of uniform, reproducible fragments between replicate PCRs. PCR thermal cycler was programmed for fi rst denaturation step at 94 °C for 1 min, followed by 40 cycles of 1 min at 94 °C, 1 min at 36 °C, and 2 min at 72 °C. Th e PCR tubes were kept at 72 °C for 7 min and then held at 4 °C until the tubes were removed.
Gel Electrophoresis
All visible and unambiguously scorable fragments were recorded. Th e fragments that were repeatedly present in one bulk and absent in the other were scored as polymorphic fragments. Polymorphic primers were used to verify the linkage of marker with the trait. RAPD fragments were separated electrophoretically on 0.9% agarose gels in 1× TBE buff er, stained with ethidium bromide, and photographed on a UV transilluminator using a digital camera. DNA from each plant was amplifi ed with the same primer 3 times, and the banding patterns were compared.
Data analysis for PCR-RAPD
RAPDs behave as dominant markers (Hyman et al. 2003), thus they tend to be used with a bistate
Exploration of genotype specifi c fi ngerprinting of Nigella sativa L. using RAPD markers
572
Tab
le 1
. Lis
t o
f P
CR
-RA
PD
pri
mer
s u
sed
fo
r p
reli
min
ary
DN
A s
cree
nin
g an
d fi
nge
rpri
nti
ng
in 3
2 a
cces
sio
ns
of
Nig
ella
sat
iva
L. g
erm
pla
sm.
Pri
mer
s S
equ
ence
(5
’ to
3’)
PC
R r
esp
on
se
Pri
mer
sS
equ
ence
(5
’ to
3’)
PC
R
resp
on
seP
rim
ers
Seq
uen
ce (
5’ t
o 3
’)P
CR
resp
on
se
OPA
-15
’-C
AG
GC
CC
TT
C-3
’+
OP
C-2
75
’-G
AG
GA
CG
TT
AA
A-3
’–
OP
S-3
5’-
CA
GA
GG
TC
CC
-3’
+
OPA
-25
’-T
GC
CG
AG
CT
G-3
’+
OP
C-4
15
’-C
AG
AC
AG
GG
TA
T-3
’–
OP
S-4
5’-
CA
CC
CC
CT
TG
-3’
+
OPA
-35
’-A
GT
CA
GC
CA
C-3
’+
OP
C-4
45
’-G
GC
AA
CA
TA
GT
A-3
’–
OP
S-5
5’-
TT
TG
GG
GC
CT
-3’
+
OPA
-45
’-A
AT
CG
GG
CT
G-3
’+
OP
C-6
75
’-C
CA
AG
AT
CC
AT
T-3
’–
OP
S-6
5’-
GA
TA
CC
TC
GG
-3’
+/–
OPA
-55
’-A
GG
GG
TC
TT
G-3
’–
OP
C-7
15
’-T
TC
AA
CA
TC
GA
C-3
’–
OP
S-7
5’-
TC
CG
AT
GC
TG
-3’
+/–
OPA
-65
’-G
GT
CC
CT
GA
C-3
’+
/–O
PC
-75
5’-
GA
TG
GT
GA
CG
AA
-3’
–O
PS-
85
’-T
TC
AG
GG
TG
G-3
’–
OPA
-75
’-G
AA
AC
GG
GT
G-3
’–
OP
C-8
55
’-A
CT
TT
GA
CA
GC
G-3
’–
OP
S-9
5’-
TC
CT
GG
TC
CC
-3’
–
OPA
-85
’-G
TG
AC
GT
AG
G-3
’+
OP
C-9
25
’-A
TC
GA
CG
GA
GA
A-3
’–
OP
S-1
05
’-A
CC
GT
TC
CA
G-3
’+
OPA
-95
’-G
GG
TA
AC
GC
C-3
’+
OP
C-9
65
’-A
CC
AA
GA
AA
GG
G-3
’–
OP
Z-1
5’-
TC
TG
TG
CC
AC
-3’
+/–
OPA
-10
5’-
GT
GA
TC
GC
AG
-3’
+O
PC
-97
5’-
AA
GA
CG
GT
GG
TA
-3’
–O
PZ
-25
’-C
CT
AC
GG
GG
A-3
’+
/–
OPA
-11
5’-
CA
AT
CG
CC
GT
-3’
+/–
OP
C-9
85
’-A
CC
AA
CG
TG
TA
C-3
’–
OP
Z-3
5’-
CA
GC
AC
CG
CA
-3’
+/–
OPA
-12
5’-
TC
GG
CG
AT
AG
-3’
+/–
OP
F-2
25
’-A
AG
AT
CA
AA
GA
C-3
’+
OP
Z-4
5’-
AG
GC
TG
TG
CT
-3’
–
OPA
-13
5’-
CA
GC
AC
CC
AC
-3’
+/–
OP
N-1
5’-
CT
CA
CG
TT
GG
-3’-
3’
+/–
OP
Z-5
5’-
TC
CC
AT
GC
TG
-3’
+/–
OPA
-14
5’-
TC
TG
TG
CT
GG
-3’
–O
PN
-25
’-A
CC
AG
GG
GC
A-3
’+
OP
Z-6
5’-
GT
GC
CG
TT
CA
-3’
+/–
OPA
-15
5’-
TT
CC
GA
AC
CC
-3’
–O
PN
-35
’-G
GT
AC
TC
CC
C-3
’–
OP
Z-7
5’-
CC
AG
GA
GG
AC
-3’
–
OPA
-16
5’-
AG
CC
AG
CG
AA
-3’
–O
PN
-45
’-G
AC
CG
AC
CC
A-3
’–
OP
Z-8
5’-
GG
GT
GG
GT
AA
-3’
+
OPA
-17
5’-
GA
CC
GC
TT
GT
-3’
–O
PN
-55
’-A
CT
GA
AC
GC
C-3
’–
OP
Z-9
5’-
CA
CC
CC
AG
TC
-3’
+
OPA
-18
5’-
AG
GT
GA
CC
GT
-3’
–O
PN
-65
’-G
AG
AC
GC
AC
A-3
’–
OP
Z-1
05
’-C
CG
AC
AA
AC
C-3
’–
OPA
-19
5’-
CA
AA
CG
TC
GG
-3’
+/–
OP
S-1
5’-
CT
AC
TG
CG
CT
-3’
–
OPA
-20
5’-
GT
TG
CG
AT
CC
-3’
–O
PS-
25
’-C
CT
CT
GA
CT
G-3
’–
58
pri
mer
s w
ith
10
an
d 1
2 b
ase
pai
rs; p
rim
ers,
oli
go-n
ucl
eoti
des
; + r
epre
sen
ts a
mp
lifi
cati
on
of
pri
mer
; – n
o a
mp
lifi
cati
on
an
d +
/– r
epre
sen
ts l
ow
am
pli
fi ca
tio
n r
esp
on
se; o
nly
amp
lifi
ed p
rim
ers
wer
e u
sed
in
th
e p
rese
nt
stu
dy.
M. S. IQBAL, S. NADEEM, S. MEHBOOB, A. GHAFOOR, M. I. RAJOKA, A. S. QURESHI, B. NIAZ
573
(present-absent) type of scoring. Photographs from
ethidium bromide stained polyacrylamide gels were
used to score the data for RAPD analysis. Each
DNA fragment amplifi ed by a given primer was
treated as a unit character and the RAPD fragments
were scored as present (1) or absent (0) for each of
the primer-accession combinations. Since DNA
samples consisted of a bulk sample of DNA extracted
from individual plants, a low intensity for any
particular fragment may be explained by the lesser
representation of that specifi c sequence in the bulk
sample of DNA. Th erefore, the intensity of the bands
was not considered and the fragments with identical
mobility were scored as identical fragments. Only
major bands were scored and faint bands were ignored.
Th e molecular size of the amplifi cation products was
calculated from a standard curve based on the known
size of DNA fragments of a marker X 174/Hae III
digest. Th e presence and absence of the bands was
scored in a binary data matrix. Pair-wise comparisons
of the accessions based on the presence or absence of
unique and shared amplifi cation products were used
to generate similarity coeffi cients. DNA bands shared
by all the accessions were excluded from the data
analysis since they are not informative (Hyman et al.
2003). Th e resulting similarity coeffi cients were used
to evaluate the relationships among the accessions
with cluster analysis using an unweighted pair-
group method with arithmetic averages (UPGMA)
and then plotted in the form of a dendrogram using
STATISTICA version 6.0 for Windows. Genetic
similarities were estimated using Nei’s standard
(1973) genetic distances as Euclidean distances also
using STATISTICA.
Results
Fift y-eight 10 to 12 mer primers were tested on
4 accessions (PK-020545, PK-020561, PK-020567,
and PK-020576) for preliminary DNA amplifi cation
and screening. Out of 58 primers, 28 revealed
amplifi cation for present material (Figure 1 and 2).
Out of amplifi ed primers, 15 exhibited polymorphism
and hence were used for fi ngerprinting of Nigella
sativa L. germplasm for further studies. Some of the
primers revealed characteristic fragments for some
accessions that were not produced in others. Some of
the primers generated several markers and were able
to show high genetic diversity, while others produced
few markers and suggested little variability. Fift een
primers generated 249 reproducible and scorable
amplifi cation products across all the accessions, out
of which 164 (66%) fragments were polymorphic
in one or other of the 32 accessions. Cluster pattern
developed for PCR-RAPD markers divided into
8 clusters on 75% Euclidean distances (Figure 3).
Accession (PK-020646) was present in cluster-1
and 2 accessions (PK-020631 and PK-020576) were
present in cluster-2, while cluster-3 consisted of 9
accessions. Cluster-4 consisted of 2 accessions, and
C-5 (6), C-6 (5), and C-7 consisted of 7 accessions of
wide genetic base.
0.5KB
10.0KB
8.0KB
6.0KB
5.0KB4.0KB3.0KB2.0KB1.5KB
1.0KB
Figure 1. PCR-RAPD polymorphism generated by OPS-5, OPA-9, OPA-3, OPA-8, and
OPS-4 in Nigella sativa L. germplasm. One kb DNA ladder visualized by ethidium
bromide staining on a 0.9% TAE agarose gel. Th e digested DNA includes fragments
ranging from 0.5 to 10.0 kilobases.
Exploration of genotype specifi c fi ngerprinting of Nigella sativa L. using RAPD markers
574
In the present study, the proportions of common bands were low (34%). Based on unweighted pair group method (UPGMA), 2 main groups and 7 sub-groups were distinguishable in the present material. Many primers generated accession specifi c
amplifi cation products and thus preliminary mapping (genetic maps) of Nigella sativa L. is suggested. Th e size of amplifi cation products scored in 1.4% agarose gels ranged between 0.5 and 10.0 kb.
0.5KB
10.0KB
1.0KB
1.5KB
8.0KB
6.0KB
4.0KB
5.0KB
3.0KB2.0KB
Figure 2. PCR-RAPD polymorphism generated by OPA-1, OPN-2, OPF-22, OPZ-8, and OPA-10 in Nigella
sativa L. germplasm. One kb DNA ladder visualized by ethidium bromide staining on a 0.9% TAE
agarose gel. Th e digested DNA includes fragments ranging from 0.5 to 10.0 kilobases.
0.5
1.0
1.5
2.0
2.5
3.0
3.5
C-1
(1
)
C- 2
(2
)
C-3
(9
)
C-4
(2
)
C-5
(6
)
C-6
(5
)
C-7
(7
)
PK
-02
06
46
P
K-0
20
63
1
PK
-02
05
76
P
K-0
20
87
8
PK
-02
08
76
PK
-02
08
77
P
K-0
20
87
5
PK
-02
06
99
PK
-02
07
29
PK
-02
07
42
P
K-0
20
78
0
PK
-02
07
66
P
K-0
20
78
1
PK
-02
05
61
PK
-02
05
67
P
K-0
20
58
5
PK
-02
06
20
PK
-02
05
92
P
K-0
20
60
9
PK
-02
08
74
PK
-02
06
62
P
K-0
20
66
3
PK
-02
08
67
PK
-02
07
20
PK
-02
08
71
P
K-0
20
86
8
PK
-02
08
72
P
K-0
20
87
3
PK
-02
07
83
PK
-02
05
45
P
K-0
20
65
4
PK
-02
07
49
Eu
clid
ean
dis
tan
ce
Figure 3. Cluster pattern for PCR-RAPD markers constructed by UPGMA of 32 genotypes of
Nigella sativa L. germplasm.
M. S. IQBAL, S. NADEEM, S. MEHBOOB, A. GHAFOOR, M. I. RAJOKA, A. S. QURESHI, B. NIAZ
575
Twenty-seven bands were recorded in 13 genotypes,
presented in Table 2. Th ese bands were marker bands
only located in these genotypes and there was no
duplication of the genotype for a particular primer.
In the Figure 4, the particular genotype specifi c bands
are displayed and compared with molecular markers
of 1 kb of DNA ladder. Th e bands were clear in
visibility and sharpness. Th e highest genetic distances
were present between accession PK-020576 (3.74)
and accession PK-020875, followed by 3.66 between
PK-020567 and PK-020877. Th e shortest distances
were recorded between PK-020742 (1.00) and PK-
Table 2. Genotype specifi c PCR-RAPD markers with band size detected in 32 genotypes of Nigella sativa L. germplasm.
No. Primers Sequences Genotypes Band size (kb)
1 OPS-3 5-CAGAGGTCCC-3
(PK-020620) 1.5
(PK-020631) 10.0, 4.5, 4.0
2 OPA-1 5-CAGGCCCTTC-3
(PK-020567) 0.5
(PK-020631) 2.0
(PK-020868) 1.0
3 OPA-9 5-GGGTAACGCC-3
(PK-020592) 8.0, 5.0
(PK-020631) 3.0
(PK-020646) 5.5
(PK-020663) 7.0
(PK-020729) 10.0
4 OPZ-8 5-GGGTGGGTAA-3
(PK-020631) 7.0
(PK-020646) 0.5, 1.0
(PK-020874) 1.3
5 OPA-3 5-AGTCAGCCAC-3
(PK-020585) 10.0
(PK-020620) 9.0
(PK-020631) 4.0, 2.0
(PK-020646) 2.3
6 OPF-22 5-AAGATCAAAGAC-3
(PK-020545) 7.0
7 OPA-8 5-GTGACGTAGG-3
(PK-020576) 1.0
(PK-020742) 0.7
8 OPN-2 5-ACCAGGGGCA-3
(PK-020576) 5.0
(PK-020592) 6.0
9 OPS-05 5-TTTGGGGCCT-3
(PK-020742) 9.0
(PK-020749) 0.5
(PK-020780) 2.0
Exploration of genotype specifi c fi ngerprinting of Nigella sativa L. using RAPD markers
576
020780, expressing linkage between them. High and
short genetic distances recorded between diff erent
accessions might be helpful in the construction of
genetic maps aft er further studies.
Discussion
As a complement to morphological, biochemical,
ecological, and genetic information, molecular
markers can contribute greatly to the use of genetic
diversity through the descriptive information they
provide on the structure of gene pools and accessions.
Th e use of RAPD techniques for germplasm
characterization facilitates the conservation and
utilization of plant genetic resources, permitting
the identifi cation of unique accessions or sources of
genetically diverse germplasm (Brown-Guedira et
al. 2001; Li et al. 2001; Vieira et al. 2001). Th e ability
of this method to distinguish between taxa also has
useful implications in botanical quality analysis
(Kapteyn and Simon 2002).
Th e technique was found useful for
characterization of present germplasm, and its ability
to sample any portion of the genome of Nigella sativa
L. could help to study markers on all the linkage
groups, genotypes distinctness, and classifi cation of
accessions into specifi c groups. Th e RAPD meets all
these requirements, although it suff ers from other
serious drawbacks such as the dominant nature
of markers, non-reproducibility of patterns, and
diffi culty in establishing homology of amplifi cation
products with similar molecular weights, which
reduce the quality of information obtained (Korzun
et al. 2001).
In the present study, RAPD markers were
used to determine genetic diversity in Nigella
sativa L. germplasm and it was concluded that
the technique was an eff ective tool in identifying
Nigella sativa L. germplasm and determining their
genetic relationships. Th e present study revealed
that working germplasm is not suffi cient to build a
comprehensive database for Nigella sativa L. Th e
information generated, however, will be helpful to
expand the scope of further research by adding new
accessions not only from local growing conditions
but also from international markets and gene banks.
As some primers revealed characteristic fragments
for some accessions that were not produced in
others, these accessions could be utilized as new
breeding material for local needs. Some of the
primers generated several markers and were able to
show high genetic diversity, while others produced
few markers and detected little variability. Fift een
primers generated 249 reproducible and scorble
amplifi cation products across all the accessions, out
of which 164 (66%) fragments were polymorphic
Mo
lecu
lar
Mar
ker
PK
-020
780
PK
-020
631
PK
-020
646
PK
-020
585
PK
-020
620
PK
-020
620
PK
-020
631
PK
-020
567
PK
-020
631
PK
-020
868
PK
-020
592
PK
-020
631
PK
-020
646
PK
-020
663
PK
-020
631
PK
-020
646
PK
-020
874
PK
-020
545
PK
-020
576
PK
-020
742
PK
-020
576
PK
-020
592
PK
-020
742
PK
-020
749
A-3 A-3 S-3 A-1 A-1A-1 A-9 A-9 Z8 A8Z8 F-22 N-2 S-5
10.0KB
6.0KB8.0KB
5.0KB
4.0KB
2.0KB3.0KB
1.5KB
1.0KB
0.5KB
Figure 4. PCR based RAPD molecular markers as genotype specifi c bands explored in 32 genotypes of Nigella
sativa L. germplasm. DNA ladder 1 kb obtained from Sigma Co. USA ranged from 0.5 to 10.0 kb.
M. S. IQBAL, S. NADEEM, S. MEHBOOB, A. GHAFOOR, M. I. RAJOKA, A. S. QURESHI, B. NIAZ
577
in one or other accessions under study. Th e level of polymorphism varied with diff erent primers among various accessions. Th e RAPDs have also been used to estimate genetic diversity among crop germplasm (Kresovich et al. 1992; Farnham 1996), for plant breeding and seed testing programs (Jianhua et al. 1996) and for tagging agronomical traits (Joshi and Nguyen 1993). Halward et al. (1991) used molecular markers in peanut but did not observe signifi cant level of polymorphism. Th is could have been due to the material and primers used in these experiments, as selection of primer in such studies is very important.
In 13 genotypes, 27 bands of diff erent mass (kilobases) were recorded that were considered specifi c to that genotype. Th ese specifi c/cloned PCR-RAPD products can be used as molecular markers for germplasm identifi cation and resource protection of Nigella sativa L. genotypes. Moreover, these products could be used for sequencing that might be successfully converted into SCAR (sequence characterized amplifi cation region) markers. Th e developed RAPD-DNA fi ngerprinting and specifi c genetic markers could provide useful ways
for the identifi cation, classifi cation, and resource
protection of the Nigella sativa L. genotypes, being
one of the important medicinal herbs listed among
underutilized species of enormous economic
importance. Th e information could be use to start
research for the establishment of medicinal plant
genomic databases.
Diverse germplasm is vital for any crop
improvement program and we have preserved the
evaluated accessions under the same accession
numbers in the gene bank at 15 °C with <40% relative
humidity for coming years. Th ese are available for
research and development work to researchers. Th e
identifi ed accessions of Nigella sativa L. could be
used as raw material for herbal, pharmaceutical,
neutraceutical, and cosmetic industry including
edible oils to enhance farmers’ productivity and
income. Collection missions are proposed to be
arranged within the areas where genetic diversity
has not been yet gathered. Probably there are some
variety-specifi c RAPD/RFLPs in Nigella sativa L.
useful for fi ngerprinting purposes.
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