Molecular Biology Today (2002) 3(1): 1-10.

ISSR-, DAMD- AND RAPD-PCR Profiles of Neem 1

Assessment of Profile Variations Amongst Provenances

of Neem Using Single-Primer-Amplification Reaction
(SPAR) Techniques#

S. A. Ranade* and Nuzhat Farooqui1

Plant Molecular Biology Division, National Botanical
Research Institute, Rana Pratap Marg, Lucknow 226 001
(U.P.) India
1Present Address: 1423 Missisauga Blvd., Ontario, Canada
L5A 4A5

Abstract
The Single Primer Amplification Reaction (SPAR)
methodology in conjunction with Simple Sequence
Repeat (SSR), minisatellite sequences and arbitrary
sequence primers are useful techniques for
determination and analysis of genetic diversity in
plants. These techniques were applied for the first time
to our knowledge in case of neem provenances to
reveal genetic relatedness between them on the basis
of the discrete profiles produced. However, our results
indicate that the neem provenances have greater-thanexpected similarities in their inter-SSR and interminisatellite regions. These conclusions are in fact
similar to those we have made earlier in case of neem
provenances using only the RAPD profiles. Thus neem
appears to have spread in India starting from only a
few groups of the original founder neem plants
introduced long ago.
Introduction
Simple Sequence Repeats (SSR) or microsatellites are
tandem repeats of di-, tri-, tetra- and penta-nucleotides and
are ubiquitous in eukaryotic genomes (Tautz, 1989; Weber
and May, 1989; Weising et al., 1989; Condit and Hubbell,
1991). Microsatellite primer (as such or anchored with 1-4
bases) anneals to an SSR region and amplifies regions
between adjacent SSR (hence called as inter-simple
sequence repeat region; ISSR region). These ISSR regions
revealing polymorphism have been proposed (Tsumura et
al., 1996) as a new source of genetic markers that can
Abbreviations
DAMD: Direct amplification of minisatellite DNA
ISSR: Inter simple sequence repeat
MP: Microsatellite primed
PCR: Polymerase chain reaction
RAPD: Random amplified polymorphic DNA
SPAR: Single primer amplification reaction

*For correspondence. Email shirishranade@yahoo.com;

Fax. (91) 522 205836, 205839.
#
NBRI communication number 504

2002 Caister Academic Press

overcome many of the technical limitations of RFLP and

RAPD analyses. To develop SSR markers, however, a
strategy of constructing genomic library, screening by
hybridization with SSR probes and sequencing positively
hybridized clones is generally used (Condit and Hubbell,
1991; Wu and Tanksley, 1993; Lavi et al., 1994). Another
strategy that has been used is to search for SSR in public
sequence database (Wang et al., 1994). Primers are then
designed from the sequence motifs to amplify
microsatellites (Smulders et al. , 1997). The primary
disadvantage, however, of such approaches for
development of SSR markers is the cost and research effort
required to clone and sequence the SSR containing DNA
fragments from plant species such as neem where no
nucleotide sequence data exist or are inadequate to identify
SSRs.
The minisatellites, also called Hyper Variable Repeats
(HVR) or Variable Number of Tandem Repeats (VNTR),
are tandem repeats of a 10-60bp long DNA sequence motif.
Variations in the number of tandem repeats at a minisatellite
locus are considered to be the source of the observed
polymorphism in a variety of organisms. Todate, a number
of minisatellite sequences have been identified in humans,
animals and plant species (Jeffreys et al., 1985; Brown
and Tanksley 1993; Winberg et al.,1993; Tourmente et
al.,1994). Minisatellite loci are inherited in a Mendelian
fashion and are dispersed throughout the genome in plants
(Brown and Tanksley, 1993; Zhou and Gustafson, 1995).
Most of the minisatellites share common motifs known as
core sequences (Nakamura et al., 1987). Therefore,
minisatellites from one source can reveal fingerprint
patterns in heterologous systems when either the entire
minisatellite or its core sequence is used as a probe in Sblot hybridization experiments to reveal unique profiles
in the target systems. Human and bacteriophage M13
sequences have frequently been employed to reveal
fingerprint profiles in plants (Dallas, 1988; Rogstad et al.,
1988; Nybom and Schaal, 1990; Nybom et al., 1990; Brown
and Tanksley, 1993). The S-blot hybridization methods for
DNA fingerprinting are in general, elaborate and often
require radioactive nucleotides. Consequently, these
techniques have not been used on a large-scale. The
increasing applications of PCR have influenced DNA
fingerprinting also. Primers derived from minisatellite
regions of the genome have been used in PCR to detect
fingerprints in several organisms (Weber and May, 1989;
Tourmente et al., 1994; Zhou and Gustafson, 1995).
However, development of primers for amplifying
minisatellites in a genome requires a study of the
organization of the minisatellites in that genome. Such an
approach can not be used in case of plants where genome
sequence data are scanty or lacking.

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2 Ranade and Farooqui

Table 1. List of neem provenances selected for SPAR studies

Sample No.

State / Country

Collected from

For MP-PCR studies

1
NBRI Botanical Garden, Lucknow
5
Banthra Research Centre, Lucknow
8
NBRI Botanical Garden, Lucknow
12
Kanpur
15
Jhansi
18
Bahraich
20
Gorakhpur
23
Haldwani
34
Ranichaura
35
Kanpur
36
Kanpur
25
Jabalpur
33
Saugar
28
Ayodhya
26
Jodhpur

Provenances

Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Madhya Pradesh
Madhya Pradesh
West Bengal
Rajasthan

NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow

For DAMD-PCR studies

37
Kanpur 46
38
Kanpur
40
Mathura
41
Jabalpur
42
Hoshangabad
43
Indore
44
Katni
45
Bilaspur
47
Raipur
48
Sohangi
49
Maihar
50
Kutalia
51
Govindgarh
52
Ranchi
53
Muzzafarpur
54
Gurgaon
55
Ladpur Kota
56
Bikaner
57
Jaisalmer
58
Sikar
59
Sawai Madhopur
60
Pali
61
Variant
62
Rajkot
66
Amravati
67
Sholapur
68
Ravinagar
69
Pune
70
Mulug
71
Bankura
72
N.Delhi
73
Nepal
74
Pakistan 1
75
Pakistan 2
76
Poi Tao
77
Uthai Thani
78
Ban Noug Hai
79
Khao Luang
80
Ban Huey Sai
8
Lucknow
34
Ranichaura
16
Jhansi
19
Bahraich
21
Gorakhpur
25
Jabalpur
33
Saugar
28
Ayodhya

Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Madhya Pradesh
Bihar
Bihar
Haryana
Rajasthan
Rajasthan
Rajasthan
Rajasthan
Rajasthan
Rajasthan
Rajasthan
Gujrat
Maharashtra
Maharashtra
Maharashtra
Maharashtra
Karnataka
West Bengal
Delhi
Nepal
Pakistan
Pakistan
Thailand
Thailand
Thailand
Thailand
Thailand
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Madhya Pradesh
Madhya Pradesh
West Bengal

AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow

For RAPD-PCR studies

1
NBRI Botanical Garden, Lucknow
5
Banthra Research Centre, Lucknow
8
NBRI Botanical Garden, Lucknow
35
Kanpur
36
Kanpur
37
Kanpur 46
38
Kanpur
74
Pakistan 1
75
Pakistan 2
76
Poi Tao
77
Uthai Thani
78
Ban Noug Hai
79
Khao Luang
80
Ban Huey Sai

Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Uttar Pradesh
Pakistan
Pakistan
Thailand
Thailand
Thailand
Thailand
Thailand

NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
NBRI, Lucknow
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur
AFRI, Jodhpur

ISSR-, DAMD- AND RAPD-PCR Profiles of Neem 3

To overcome these limitations PCR amplification was

carried out using a single primer composed of repeat
sequence and this strategy was known as Single Primer
Amplification Reaction (SPAR, Gupta et al., 1994; Weising
et al., 1995). When mini- or micro-satellite sequences are
individually used as primer for amplification, they almost
invariably show extensive polymorphism due to site-specific
length variation in the inter-repeat region. As a
consequence different number of bands (markers) are
produced. In case of microsatellite primers for SPAR, the
resulting pattern of amplified DNA corresponds to the
regions between two successive SSRs (Inter Simple
Sequence Repeat-PCR; ISSR-PCR). The SPAR approach
to minisatellite analysis has been described as Direct
Amplification of Minisatellite-region DNA (DAMD), which
directs the amplification to regions rich in minisatellite
repeats by using the core sequence of minisatellites as
single primer (Heath et al., 1993; Zhou et al., 1997).
The PCR carried out with arbitrary sequence primers
(RAPD), may actually be described as the generic
precursor of SPAR since in RAPD also a single primer but
of short arbitrary sequence was used. It (RAPD) is a
powerful technique and has been extensively employed
ever since two groups working independently developed it
simultaneously (Welsh and McClelland 1990, Williams et
al., 1990). We have earlier used this technique to score
genetic variation amongst 34 provenances of neem
(Farooqui et al., 1998). Similarly, the technique was also
used for detecting inter- and intra-species variation
amongst Prosopis accessions and species (Goswami and
Ranade, 1999).
In case of neem, no prior sequence information on
the genome is available even though it is an ancient and

Table 2. The SSR sequences, minisatellite core sequences and the arbitrary
sequence decamers used as primers in amplification reactions.
S.No.

Primer Name

Sequence (5'-3'), Length

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15.
16.
17.
18.
19.
20.
21.

TATC-6
TGTC-4
GATA-6
GACA-4
GTG-5
CAC-6
CTT-5
GAA-G
CAC-5
GAA-6
TG-10
AT-10
AAAT-4
AATT-4
33.6a
HBV(5)b
HVR(-)c
OP-F02
OP-F03
OP-U10
OP-U20

(TATC)6, 24 mer
(TGTC)4TGT, 19 mer
(GATA)6G, 25 mer
(GACA)4, 16 mer
(GTG)5G, 16 mer
(CAC)6C, 19 mer
(CTT)5C, 16 mer
(GAA)6G, 19 mer
(CAC)5, 15 mer
(GAA)6, 18 mer
(TG)10, 20 mer
(AT)10, 20 mer
(AAAT)4, 16 mer
(AATT)4, 16 mer
GGAGGTTTTCA, 11 mer
GGTGTAGAGAGAGGGGT, 17 mer
CCTCCTCCCTCCT, 13 mer
GAGGATCCCT, 10 mer
CCTGATCACC, 10 mer
ACCTCGGCAC, 10 mer
ACAGCCCCCA, 10 mer

a
Primer synthesized from Core sequence of 33.6 minisatellite (Jeffreys et
al., 1985)
b
Primer synthesized from core sequence of HBV minisatellite (Nakamura
et al., 1987)
c
Primer synthesized from core sequence of HVR minisatellite (Winberg et
al., 1983)

well-known tree, with multiple uses especially as a source

of numerous phytochemicals with insecticidal and insectrepellant properties, and with a wide distribution in India
and South East Asian countries. Furthermore, not much is
known about the extent and pattern of natural genetic
variation present in neem. We have, therefore, initiated
the work on the determination and analysis of DNA profile
variation amongst neem provenances using PCR
approaches. We have already obtained evidences from
RAPD profile analysis that neem provenances seemingly
have a narrow genetic base (Farooqui et al., 1998). In this
study, SPAR techniques along with RAPD were applied
for assessment of variations in DNA profiles in neem.
Results
The variability of genomes can be studied using several
different approaches. In the present study, PCR based
fingerprinting, namely, SPAR (Gupta et al., 1994), has been
used to determine genetic variability in neem. The original
method of SPAR used SSRs as primers. However, Heath
et al. (1993) had earlier described another technique
wherein a minisatellite primer was used singly in PCR. Thus
this is also another SPAR method. These two methods
should prove to be invaluable for use with all genomes
where sequence data are either scanty or lacking
altogether. RAPD is also a well-known method applicable
to all genomes irrespective of the availability of nucleotide
sequence data.

SPAR with SSR Primers

The primers used were 15-25 bases long and represented
SSRs with repetitions of di-, tri-, and tetra nucleotide motifs
(Table 2). The annealing temperatures were optimized for
each SSR-primed PCR. The results of such an optimization
are given in Table 3. Of the fourteen primers tested, 50%
produced patterns of discrete bands. Most of the oligomers
with tetranucleotide repeats either did not result in any
amplification or resulted only in a smear. The trinucleotide
repeat primers on the other hand were most effective and

Table 3. Determination of the optimum annealing temperature for the

different microsatellite or SSR primers. Td was calculated as described in
the materials and methods. The shaded areas indicate that the best profiles
were obtained at that temperature for the primer. The PCR results are
indicated as P for multibanded profiles, NP for no profiles and S for smears
or fuzzy bands.
S. No. Primer Name Td (C)
as in Table 2
1
2
3
4
5
6
7
8
9
10
11
12
13
14

TATC-6
TGTC-4
GATA-6
GACA-4
GTG-5
CAC-6
CTT-5
GAA-G
CAC-5
GAA-6
TG-10
AT-10
AAAT-4
AATT-4

60
56
64
48
54
64
44
52
50
48
60
40
32
32

Annealing temperature (C)

and Result of PCR
51,
45,
54,
38,
45,
54,
34,
42,
42,
38,
51,
30,
22,
22,

NP
NP
NP
P
P
P
NP
P
S
P
NP
NP
NP
NP

54,
48,
57,
42,
49,
57,
38,
45,
45,
42,
54,
34,
26,
26,

NP
NP
NP
P
P
P
NP
P
S+P
P
NP
NP
NP
NP

57,
52,
61,
45,
51,
61,
42,
47,
47,
45,
57,
38,
30,
30,

P
NP
S
P
P
P
NP
P
S
P
S
NP
NP
NP

4 Ranade and Farooqui

Figure 1. ISSR-PCR profiles of neem provenances using (a) primer (GAA)6G, (b) primer (CAC)5 and (c) primer (GAA)6. The products of the amplification
reactions are analyzed by electrophoresis through 2% (w/v) agarose gels in TBE buffer made according to Sambrook et al., (1989). The numbers above the
lanes correspond to the provenance sample numbers as given in Table 1. The lane marked with M contains DNA molecular weight marker ( DNA double
digested with EcoRI and HindIII enzymes). The molecular weights of some of the bands in the marker lane are given to the left of the photos. #5.14 kb (Two
bands of 5.14 and 4.97 kb), *2.02 kb (Two bands of 2.02 and 1.90 kb) and +1.58 kb (Two bands of 1.58 and 1.33 kb).

resulted in distinct amplification profiles with most of the

primers revealing broadly similar profiles across the
provenances.
DNA from 15 different provenances (Table 1) was
amplified using the primers according to optimum
conditions as described above. In experiments with (GAA)6,
(GAA)6G and (CAC)5 most informative patterns were
produced at 45C which is the optimal annealing
temperature for these primers. Primers with repeat motifs
(TGTC), (CTT), (AT), (AAAT) and (AATT) did not produce
any profiles at three different annealing temperatures tested
(Table 3). The profiles obtained after amplification of DNAs
of 15 provenances with (GAA)6G, (CAC)5 and (GAA)6 are
depicted in Figure 1a to 1c respectively. The profiles
obtained were mostly similar between provenances.
However, the numbers of amplification products
(corresponding to ISSR-regions) were different between
the provenances while there were no obvious bands of

different sizes (polymorphic) that were present. The number

of ISSR-regions amplified ranged from 3 in case of some
of the provenances with (GAA)6G as primer to as many as
10-14 in case of (CAC)5 and (GAA)6 as primers. In general,
the products obtained were of low molecular weight, in the
range of 1200-180bp with (GAA)6G primer and 2100-220bp
with (CAC)5 and (GAA)6 primers. Provenance number 12
in case of (GAA)6G and provenance number 25 in case of
(GAA)6 primer did not result in any profile (Figure 1a, 1c
respectively). However, the same DNAs did reveal good
profiles with the other primers tested.

SPAR with Minisatellite Primers

DAMD PCR was carried out essentially according to Zhou
et al. (1997) using Idaho Air Thermal Cycler. There were
two to ten clearly distinguishable bands in the different
samples of neem, with distinctly polymorphic banding
patterns, after amplification with the 33.6 minisatellite primer

ISSR-, DAMD- AND RAPD-PCR Profiles of Neem 5

Figure 2. DAMD-PCR profiles of neem provenances using 33.6 minisatellite core primer. The products of the amplification reactions are analyzed by
electrophoresis through 2% (w/v) agarose gels in TBE buffer made according to Sambrook et al., (1989). The neem provenances are identified with numbers
corresponding to the list in Table 1, above the lanes, while lanes marked with M contain DNA molecular weight marker ( DNA double digested with EcoRI
and HindIII enzymes). The molecular weights of some of the bands in the marker lane are given to the left of the photos. #5.14 kb (Two bands of 5.14 and 4.97
kb)

(Figure 2). In the case of profiles obtained with 33.6

minisatellite core sequence primer, provenance numbers
24, 29 and 63-65 failed to amplify. Therefore, these
provenances were not considered for the UPGMA analysis.
Though no individual specific profile was obtained, there
were several small groups of samples with similar types of
profile (Figure 3) even in the UPGMA dendrogram. Though
the eight exotic provenances (five from Thailand, two from
Pakistan and one from Nepal) grouped together, these,

however, did not separate out as an outgroup. The

amplification products were in the range of 5600-180bp.
With HVR(-) only smears were obtained in most cases,
while with HBV5 primer, the profiles were not very distinct
since only faint bands were visible along with background
smear. These two profiles (with HBV5 and HVR primers)
did not improve even after varying different reaction
parameters and annealing stringency conditions (data not
shown).

Figure 3. UPGMA dendrogram of the neem provenances analyzed by DAMD-PCR using 33.6 minisatellite core primer. The scale on the top indicates
distances while the numbers to the right identify the neem provenances according to Table 1. The DAMD-PCR band data was analyzed pairwise for
Jaccards similarity coefficients and distances thereof. The distances were then used to construct a dendrogram according to Sokal and Sneath (1963).

6 Ranade and Farooqui

Figure 4. RAPD-PCR profiles of the neem provenances using primers (a) OP-F02, (b) OP-F03, (c) OP-U10 and (d) OP-U20. The lane numbers correspond
to the sample numbers as given in Table 1. Lane marked M contains DNA molecular weight marker ( DNA double digested with EcoRI and HindIII enzymes).
The molecular weights of some of the bands in the marker lane are given to the left of the photos. *2.02 kb (Two bands of 2.02 and 1.90 kb) and +1.58 kb
(Two bands of 1.58 and 1.33 kb).

RAPD-PCR
The RAPD-PCR profiles in case of the 14 provenances
obtained with the four primers OP-F02, OP-F03, OP-U10
and OP-U20 are given in Figure 4 a, b, c, d respectively.
With primers OP-F02 and OP-F03, some of the bands were
observed in all 14 provenances (indicated with an arrow,
Figure 4a,b) indicating that these had presumably been
amplified from conserved genomic regions. Similarly with
primers OP-F02 and OP-U20 all the Thailand provenances
showed the presence of bands that were not present in
any other provenance (indicated with an arrowhead, Figure
4a,d).

Discussion

ISSR-PCR
Inter Simple Sequence Repeat markers involves PCR
amplification of DNA using a single primer composed of
microsatellite sequences, sometimes anchored at the 3'
or 5' end by 1 to 4 nucleotides. These primers target the
simple sequence repeats and amplify the intervening region
between the two SSRs in opposite orientations. This,
technique does not require prior knowledge of the genome,
since these SSRs are abundantly present in the plant
genome in many copies of varying repeat units

ISSR-, DAMD- AND RAPD-PCR Profiles of Neem 7

(Langercrantz et al., 1993). Due to their varying repeat

numbers at a given locus, the elements frequently change
their length by slipped strand mispairing, the surrounding
single copy sequence are normally not affected and
therefore provide a valuable source of polymorphism for
identification of species and cultivars (Tsumura et al., 1996;
Fang and Roose, 1997). These have been used to assess
genetic diversity even in inbred lines of dent corn and
popcorn (Kantety et al., 1995). ISSR polymorphic DNA was
evaluated for its applicability as a genetic marker system
and compared with the other commonly used markers as
RFLPs and RAPDs by Nagaoka and Ogihara (1997), in
case of wheat. The genetic relationships of wheat
accessions estimated by ISSR markers were identical to
those inferred by RFLP and RAPD markers, indicating the
reliability of ISSR markers for the estimation of genotypes.
The potential of ISSR-PCR for fingerprinting purpose was
recently evaluated in case of potato where only four
microsatellite primers were found to be sufficient for
complete diagnosis of all the cultivars (Prevost and
Wilkinson, 1999).
In the present study, the SSR primers were annealed
at different temperatures that were between 2C to 10C
lower than the Td of the primer, so as to determine the
optimum temperature for a particular microsatellite. Of the
14 primers tested, only 7 resulted in patterns. This is similar
to the situation in case of Citrus ISSR markers wherein
out of 46 primers screened, only 22 resulted in profiles
while the rest either did not produce any profiles or resulted
in a smear of fuzzy band patterns (Fang and Roose 1997).
The trinuleotide repeat primers were most effective and
resulted in amplification profiles with most of the primers
revealing broadly similar profiles across the provenances.
The patterns were reproducible among experiments, DNA
samples and primer lots. Most of the tetranucleotide repeat
primers on the other hand were ineffective and showed
smears or fuzzy and weak band profiles. Such profiles
reflect either the abundance of these repeats in the genome
or that such patterns may be the result of amplification
due to annealing to complementary strands of the repeat
to each other to form concatamers (Jeffreys et al., 1988).
Surprisingly none of the AT rich primers resulted in any
profile. One possible reason for this could be due to lack
of AT rich repeats in the genome of neem. If this were true,
neem would apparently contrast with the reported trend of
abundance of (AT)n sequences in plants (Condit and
Hubbell, 1991; Akagi et al.,1997).
The profiles obtained with SSR primers consisted of
several closely spaced bands and no fragments that were
obviously polymorphic amongst the genotype could be
identified. This was surprising since these results indicated
that the ISSR regions were also apparently conserved at
least in length if not in sequence. This is apparently true
since the neem template DNAs pre-digested with selected
restriction endonucleases prior to use for PCR with
(GAA)6G, (GAA)6 and (CAC)5 as primers also did not reveal
any large-scale variations amongst the provenances (data
not shown). It was expected that the pre-digestion of the
DNA with the restriction endonuclease may reveal
differences across the provenances in their ISSR regions
that were amplified with the SSR primer, in the form of
distinctly polymorphic bands. Furthermore, the recent

success with ISSR-PCR in case of plants like Citrus (Fang

and Roose, 1997) and wheat (Prevost and Wilkinson, 1999)
indicated the resolution power of this technique and
therefore, we expected large-scale polymorphism to have
been revealed even amongst the small group of
provenances that we selected. These provenances in most
cases are hundreds of kilometers apart. Our results indicate
that inspite of this, ISSR regions of these provenances are
mostly similar in length and possibly in sequence too. This
can only be possible if the provenances share a greater
genetic relatedness.

DAMD-PCR
Heath et al., (1993) developed a novel technique called as
DAMD, which uses PCR to direct the amplification to
regions rich in minisatellites. In DAMD-PCR, a single primer
from a minisatellite core is used to direct PCR from the
regions rich in minisatellites. These regions may have
sequences involved in inversions between successive
minisatellites resulting in their distribution on both strands
in opposite orientations. It is to these regions that the core
primer anneals and results in a discrete profile of the intersuccessive-minisatellite region. If inversions have taken
place, then the repeats would most likely have a piece of
single copy flanking DNA inserted between them.
Therefore, DAMD could also amplify sequences formerly
adjacent to hypervariable minisatellite loci. Furthermore,
since minisatellite core sequences are conserved across
species and are larger than RAPD primers, DAMD-PCR
can be effectively carried out at relatively high stringencies,
thus yielding highly reproducible results (Heath et al., 1993).
By means of highly specific M13-PCR fingerprinting
technique profiles were determined in case of Willows
(Chong et al., 1995) while the mutant and revertant tissues
were analyzed in case of Quercus sp. by Fladung and
Ziegenhagen (1998). M13 primers have also been used to
differentiate between 32 megagametophytes, which were
investigated from seeds of a single silver fir (Abies alba)
tree (Degen et al. , 1995). PCR-primed with wheat
minisatellite core sequences yielded DNA-fingerprinting
probes in wheat (Bebeli et al., 1997). Hence the minisatellite
core primers are suggested to be universally applicable in
PCR-fingerprinting experiments enabling the genetic
differentiation of individuals.
Distinctly polymorphic banding pattern was observed
when minisatellite core sequence primer 33.6 was used
with neem DNA as template. Since the PCR reactions were
done at high stringency, DAMD-PCR yielded highly
reproducible results with all samples tested. However, no
individual specific profiles were obtained. Few groups of
provenances with similar patterns were produced, showing
similar kind of genome organization. These observations
suggested that DAMD-PCR, successfully amplified the
neem genomic DNA, producing RAPD like results for the
identification of specific region in the genome. The other
two primers HVR (-) and HBV5, did not reveal any distinct
profiles. This could be due to absence or less occurrence
of an inversion site of minisatellite region or in general a
low homology of these core sequences to neem
minisatellite families unlike in case of rice where these have
resulted in discrete profiles (Zhou et al., 1997). The samples
for DAMD-PCR study included provenances from Nepal,

8 Ranade and Farooqui

Pakistan and Thailand also. However, even these exotic

provenances did not reveal any appreciable differences
relative to the Indian neem provenances, as far as their
inter-minisatellite regions are concerned.

RAPD-PCR
The RAPD studies, whenever, carried out for inter-species
comparison, invariably resulted in clear distinction of
species enabling phylogenetic and taxonomic
interpretations (Adams and Demeke 1993; Abo-Elwafa et
al., 1995; Millan et al., 1996). Thus it was expected that
the Thailand provenances (considered to represent a
different species A.siamensis ) should be clearly
distinguished from all other provenances that represent
the species A.indica. Though in our study with RAPD
primers, we have observed amplification products specific
to Thailand neem provenances in case of a few of the
primers (Figure 4a,d), this trend was not consistent for all
the primers tested. However, Singh et al. (1999) have used
AFLP to clearly distinguish between Indian and Thailand
neem provenances on the basis of a large number of AFLP
markers. The clear distinction of Thailand provenances
from all others by AFLP technique but not by the RAPD or
SPAR techniques indicates that a larger number of markers
are required for this. This suggests that the two groups of
provenances are more similar than dissimilar. Thus even
their ISSR or inter-minisatellite regions are not so well
differentiated that the Thailand neem provenances
separate out in the dendrogram as outgroups.
The results of this SPAR study with SSR, minisatellite
and RAPD primers, carried out for the first time to our
knowledge in case of neem has thus indicated the existence
of a greater-than-expected genetic similarity amongst neem
provenances. These results agree well with our earlier
assessment of a narrow genetic base in case of neem using
only the RAPD profiles (Farooqui et al., 1998). Thus neem
appears to have spread in India starting from only a few
groups of plants. It is also possible that neem though
currently widely distributed in India, did not originate here.
If this is indeed so, neem in India must have evolved from
the original founder neem trees introduced long ago, and
this may well explain the lack of obvious genetic diversity
amongst the provenances selected for the present study.
Experimental Procedures

Plant Material
The neem provenances selected for the present study with
three types of SPAR, namely, ISSR-PCR, DAMD-PCR and
RAPD-PCR, are listed in Table 1. For ISSR-PCR, we
selected 15 provenances from geographically separated
regions. For the DAMD-PCR, however, we selected a wider
range and larger number of provenances since we
assumed that the minisatellites having longer sequence
motifs than the SSRs might not reveal any large-scale
variation. For the RAPD-PCR, we selected 14 provenances
that included five from Thailand, two from Pakistan as exotic
provenances, and three from Lucknow, four from Kanpur
as Indian provenances.

SSR, Minisatellite and RAPD Primers

Fourteen SSR primers were custom synthesized from
Rama Biotech (Secunderabad, India) and CBT (New Delhi,
India), while the core sequences of three minisatellites were
custom synthesized from Bangalore Genei, Bangalore,
India. The four RAPD primers were procured from Operon
Tech. Inc., Alameda, CA, USA. The sequences of all these
primers are given in Table 2.
DNA Isolation
The total leaf tissue DNAs from the neem plants from
various provenances were isolated as described earlier
(Farooqui et al., 1998). The samples were checked both
qualitatively as well as quantitatively by agarose gel
elctrophoresis according to Sambrook et al. (1989).
SPAR with SSR Primers
The PCR conditions as proposed by Gupta et al. (1994)
were used with minor modifications in concentration of
magnesium ions. A pilot experiment was carried out to
determine the optimum concentration. The final reaction
was carried out in 10 l volumes and contained 50 ng of
template DNA (quantitation was done by DynaQuant200
fluorometer), 10 pmoles of SSR primer, 200 M each dNTP,
3.0 mM Mg2+ ion concentration in suitable 1X assay buffer
supplied along with the enzyme and 0.5 Units of the
thermostable Taq DNA polymerase (Bangalore Genei,
Bangalore, India). The tubes were placed in the Air Thermal
Cycler (Idaho Technology, USA) for the PCR. The Air
Thermal Cycler was programmed to include predenaturation at 94C for 1 minute. This was followed by
25 cycles of denaturation at 94C for 30 s, annealing at
the optimized temperature for 30 s, and extension at 72C
for 1 min. The final cycle allowed an additional 5 min period
of extension at 72C. When all the reaction components
were standardized the annealing temperature was
optimized for each SSR primer. The optimum annealing
temperature was determined in the range of 2-10C lower
than the denaturation temperature (Td) as shown in Table
3. The denaturation temperature was calculated according
to Berger and Kimmel (1987), by adding 2C for each A or
T and 4C for each G or C in the oligomer. The reaction
products obtained after PCR were analyzed on 2% agarose
gel. The gel was stained in ethidium bromide and visualized
on Nighthawk gel documentation system (pdi Inc., USA).
Only distinct and well-separated bands were considered
for the analysis.
SPAR with Minisatellite Primers
The reaction components were similar to that as optimized
for SSR primers. The reaction mixtures contained 50 ng of
template DNA, 10 pmoles primer, 3.0 mM magnesium ions,
200 M each dNTP, 0.5 Units enzyme and 1X buffer. The
volume was made upto 10l with deionized sterile water.
The DAMD-PCR was carried out essentially according to
Zhou et al. (1997). The optimum annealing temperatures
were determined by carrying out PCR at three annealing
temperatures 47C, 50C and 55C. Of the three
temperatures, 55C annealing temperature resulted in the
clearest profile. The cycling parameters were as follows:
First cycle of 94C for 1 minute for initial denaturation of

ISSR-, DAMD- AND RAPD-PCR Profiles of Neem 9

template, then 35 cycles of incubation at 94C for 30

seconds, at annealing temperature (55C) for 1 minute and
72C for 1 minute. Last cycle allowed 5 minute at 72C for
extension. These conditions were used with all minisatellite
primers. The products obtained were electrophoresed on
2% agarose gel in 0.5X TBE buffer at 5V/cm. After
electrophoresis the gel was stained in ethidium bromide
and then visualized and photographed on gel
documentation system. Only distinct and well-separated
bands were included in the analysis.

RAPD of Neem Provenances

For the RAPD studies, a smaller set of 14 provenances
was selected from amongst the 52 used for DAMD-PCR
and ISSR-PCR. While selecting this set, we have included
multiple provenances from a given region. RAPD analysis
was carried out as described earlier (Farooqui et al., 1998),
using the primers OP-F02, OP-F03, OP-U10 and OP-U20.
The reactions were carried out in 10 l volumes and
contained 50 ng of template DNA, 10 pmoles of arbitrary
sequence decamer primer, 200 M each dNTP, 2.5 mM
Mg2+ ion concentration in suitable 1X assay buffer supplied
along with the enzyme and 0.5 Units of the thermostable
Taq DNA polymerase (Bangalore Genei, Bangalore, India).
The tubes were placed in the Air Thermal Cycler (Idaho
Technology, USA) for the PCR. The Air Thermal Cycler
was programmed to include pre-denaturation at 94C for
1 minute. This was followed by 45 cycles of denaturation
at 94C for 30 s, annealing at 35C for 30 s, and extension
at 72C for 1 min. The final cycle allowed an additional 5
min period of extension at 72C. The PCR products were
analyzed by electrophoresis through 1.0 % agarose gels
as above.
Acknowledgements
We thank the Biomass Research Center and Arid Forerst
Research Institute at Lucknow and Jodhpur respectively,
for all help with provenance selection. Financial support
as Senior Research Fellowship from the CSIR to NF and
grant-in-aid from DBT to SAR are gratefully acknowledged.
We are thankful for support and encouragement of Director,
NBRI.
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