exploration of genotype specifi c fi ngerprinting of...

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


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


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.


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.


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.


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


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.


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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