Assessment of Profile Variations Neem SPAR

ISSR-, DAMD- AND RAPD-PCR Profiles of Neem 1Molecular Biology Today (2002) 3(1): 1-10.

2002 Caister Academic Press

Assessment of Profile Variations Amongst Provenancesof Neem Using Single-Primer-Amplification Reaction(SPAR) Techniques#

*For correspondence. Email [email protected];Fax. (91) 522 205836, 205839.#NBRI communication number 504

S. A. Ranade* and Nuzhat Farooqui1

Plant Molecular Biology Division, National BotanicalResearch Institute, Rana Pratap Marg, Lucknow 226 001(U.P.) India1Present Address: 1423 Missisauga Blvd., Ontario, CanadaL5A 4A5

Abstract

The Single Primer Amplification Reaction (SPAR)methodology in conjunction with Simple SequenceRepeat (SSR), minisatellite sequences and arbitrarysequence primers are useful techniques fordetermination and analysis of genetic diversity inplants. These techniques were applied for the first timeto our knowledge in case of neem provenances toreveal genetic relatedness between them on the basisof the discrete profiles produced. However, our resultsindicate that the neem provenances have greater-than-expected similarities in their inter-SSR and inter-minisatellite regions. These conclusions are in factsimilar to those we have made earlier in case of neemprovenances using only the RAPD profiles. Thus neemappears to have spread in India starting from only afew groups of the original founder neem plantsintroduced long ago.

Introduction

Simple Sequence Repeats (SSR) or microsatellites aretandem repeats of di-, tri-, tetra- and penta-nucleotides andare ubiquitous in eukaryotic genomes (Tautz, 1989; Weberand May, 1989; Weising et al., 1989; Condit and Hubbell,1991). Microsatellite primer (as such or anchored with 1-4bases) anneals to an SSR region and amplifies regionsbetween adjacent SSR (hence called as inter-simplesequence repeat region; ISSR region). These ISSR regionsrevealing polymorphism have been proposed (Tsumura etal., 1996) as a new source of genetic markers that can

overcome many of the technical limitations of RFLP andRAPD analyses. To develop SSR markers, however, astrategy of constructing genomic library, screening byhybridization with SSR probes and sequencing positivelyhybridized clones is generally used (Condit and Hubbell,1991; Wu and Tanksley, 1993; Lavi et al., 1994). Anotherstrategy that has been used is to search for SSR in publicsequence database (Wang et al., 1994). Primers are thendesigned from the sequence motifs to amplifymicrosatellites (Smulders et al., 1997). The primarydisadvantage, however, of such approaches fordevelopment of SSR markers is the cost and research effortrequired to clone and sequence the SSR containing DNAfragments from plant species such as neem where nonucleotide sequence data exist or are inadequate to identifySSRs.

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 minisatellitelocus are considered to be the source of the observedpolymorphism in a variety of organisms. Todate, a numberof minisatellite sequences have been identified in humans,animals and plant species (Jeffreys et al., 1985; Brownand Tanksley 1993; Winberg et al.,1993; Tourmente etal.,1994). Minisatellite loci are inherited in a Mendelianfashion and are dispersed throughout the genome in plants(Brown and Tanksley, 1993; Zhou and Gustafson, 1995).Most of the minisatellites share common motifs known ascore sequences (Nakamura et al., 1987). Therefore,minisatellites from one source can reveal fingerprintpatterns in heterologous systems when either the entireminisatellite or its core sequence is used as a probe in S-blot hybridization experiments to reveal unique profilesin the target systems. Human and bacteriophage M13sequences have frequently been employed to revealfingerprint profiles in plants (Dallas, 1988; Rogstad et al.,1988; Nybom and Schaal, 1990; Nybom et al., 1990; Brownand Tanksley, 1993). The S-blot hybridization methods forDNA fingerprinting are in general, elaborate and oftenrequire radioactive nucleotides. Consequently, thesetechniques have not been used on a large-scale. Theincreasing applications of PCR have influenced DNAfingerprinting also. Primers derived from minisatelliteregions of the genome have been used in PCR to detectfingerprints in several organisms (Weber and May, 1989;Tourmente et al., 1994; Zhou and Gustafson, 1995).However, development of primers for amplifyingminisatellites in a genome requires a study of theorganization of the minisatellites in that genome. Such anapproach can not be used in case of plants where genomesequence data are scanty or lacking.

AbbreviationsDAMD: Direct amplification of minisatellite DNAISSR: Inter simple sequence repeatMP: Microsatellite primedPCR: Polymerase chain reactionRAPD: Random amplified polymorphic DNASPAR: Single primer amplification reaction

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2 Ranade and Farooqui

Table 1. List of neem provenances selected for SPAR studies

Sample No. Provenances State / Country Collected from

For MP-PCR studies1 NBRI Botanical Garden, Lucknow Uttar Pradesh NBRI, Lucknow5 Banthra Research Centre, Lucknow Uttar Pradesh NBRI, Lucknow8 NBRI Botanical Garden, Lucknow Uttar Pradesh NBRI, Lucknow12 Kanpur Uttar Pradesh NBRI, Lucknow15 Jhansi Uttar Pradesh NBRI, Lucknow18 Bahraich Uttar Pradesh NBRI, Lucknow20 Gorakhpur Uttar Pradesh NBRI, Lucknow23 Haldwani Uttar Pradesh NBRI, Lucknow34 Ranichaura Uttar Pradesh NBRI, Lucknow35 Kanpur Uttar Pradesh NBRI, Lucknow36 Kanpur Uttar Pradesh NBRI, Lucknow25 Jabalpur Madhya Pradesh NBRI, Lucknow33 Saugar Madhya Pradesh NBRI, Lucknow28 Ayodhya West Bengal NBRI, Lucknow26 Jodhpur Rajasthan NBRI, Lucknow

For DAMD-PCR studies37 Kanpur 46 Uttar Pradesh AFRI, Jodhpur38 Kanpur Uttar Pradesh AFRI, Jodhpur40 Mathura Uttar Pradesh AFRI, Jodhpur41 Jabalpur Madhya Pradesh AFRI, Jodhpur42 Hoshangabad Madhya Pradesh AFRI, Jodhpur43 Indore Madhya Pradesh AFRI, Jodhpur44 Katni Madhya Pradesh AFRI, Jodhpur45 Bilaspur Madhya Pradesh AFRI, Jodhpur47 Raipur Madhya Pradesh AFRI, Jodhpur48 Sohangi Madhya Pradesh AFRI, Jodhpur49 Maihar Madhya Pradesh AFRI, Jodhpur50 Kutalia Madhya Pradesh AFRI, Jodhpur51 Govindgarh Madhya Pradesh AFRI, Jodhpur52 Ranchi Bihar AFRI, Jodhpur53 Muzzafarpur Bihar AFRI, Jodhpur54 Gurgaon Haryana AFRI, Jodhpur55 Ladpur Kota Rajasthan AFRI, Jodhpur56 Bikaner Rajasthan AFRI, Jodhpur57 Jaisalmer Rajasthan AFRI, Jodhpur58 Sikar Rajasthan AFRI, Jodhpur59 Sawai Madhopur Rajasthan AFRI, Jodhpur60 Pali Rajasthan AFRI, Jodhpur61 Variant Rajasthan AFRI, Jodhpur62 Rajkot Gujrat AFRI, Jodhpur66 Amravati Maharashtra AFRI, Jodhpur67 Sholapur Maharashtra AFRI, Jodhpur68 Ravinagar Maharashtra AFRI, Jodhpur69 Pune Maharashtra AFRI, Jodhpur70 Mulug Karnataka AFRI, Jodhpur71 Bankura West Bengal AFRI, Jodhpur72 N.Delhi Delhi AFRI, Jodhpur73 Nepal Nepal AFRI, Jodhpur74 Pakistan 1 Pakistan AFRI, Jodhpur75 Pakistan 2 Pakistan AFRI, Jodhpur76 Poi Tao Thailand AFRI, Jodhpur77 Uthai Thani Thailand AFRI, Jodhpur78 Ban Noug Hai Thailand AFRI, Jodhpur79 Khao Luang Thailand AFRI, Jodhpur80 Ban Huey Sai Thailand AFRI, Jodhpur8 Lucknow Uttar Pradesh NBRI, Lucknow34 Ranichaura Uttar Pradesh NBRI, Lucknow16 Jhansi Uttar Pradesh NBRI, Lucknow19 Bahraich Uttar Pradesh NBRI, Lucknow21 Gorakhpur Uttar Pradesh NBRI, Lucknow25 Jabalpur Madhya Pradesh NBRI, Lucknow33 Saugar Madhya Pradesh NBRI, Lucknow28 Ayodhya West Bengal NBRI, Lucknow

For RAPD-PCR studies1 NBRI Botanical Garden, Lucknow Uttar Pradesh NBRI, Lucknow5 Banthra Research Centre, Lucknow Uttar Pradesh NBRI, Lucknow8 NBRI Botanical Garden, Lucknow Uttar Pradesh NBRI, Lucknow35 Kanpur Uttar Pradesh NBRI, Lucknow36 Kanpur Uttar Pradesh NBRI, Lucknow37 Kanpur 46 Uttar Pradesh AFRI, Jodhpur38 Kanpur Uttar Pradesh AFRI, Jodhpur74 Pakistan 1 Pakistan AFRI, Jodhpur75 Pakistan 2 Pakistan AFRI, Jodhpur76 Poi Tao Thailand AFRI, Jodhpur77 Uthai Thani Thailand AFRI, Jodhpur78 Ban Noug Hai Thailand AFRI, Jodhpur79 Khao Luang Thailand AFRI, Jodhpur80 Ban Huey Sai Thailand AFRI, Jodhpur

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

well-known tree, with multiple uses especially as a sourceof numerous phytochemicals with insecticidal and insect-repellant properties, and with a wide distribution in Indiaand South East Asian countries. Furthermore, not much isknown about the extent and pattern of natural geneticvariation present in neem. We have, therefore, initiatedthe work on the determination and analysis of DNA profilevariation amongst neem provenances using PCRapproaches. We have already obtained evidences fromRAPD profile analysis that neem provenances seeminglyhave a narrow genetic base (Farooqui et al., 1998). In thisstudy, SPAR techniques along with RAPD were appliedfor assessment of variations in DNA profiles in neem.

Results

The variability of genomes can be studied using severaldifferent approaches. In the present study, PCR basedfingerprinting, namely, SPAR (Gupta et al., 1994), has beenused to determine genetic variability in neem. The originalmethod of SPAR used SSRs as primers. However, Heathet al. (1993) had earlier described another techniquewherein a minisatellite primer was used singly in PCR. Thusthis is also another SPAR method. These two methodsshould prove to be invaluable for use with all genomeswhere sequence data are either scanty or lackingaltogether. RAPD is also a well-known method applicableto all genomes irrespective of the availability of nucleotidesequence data.

SPAR with SSR PrimersThe primers used were 15-25 bases long and representedSSRs with repetitions of di-, tri-, and tetra nucleotide motifs(Table 2). The annealing temperatures were optimized foreach SSR-primed PCR. The results of such an optimizationare given in Table 3. Of the fourteen primers tested, 50%produced patterns of discrete bands. Most of the oligomerswith tetranucleotide repeats either did not result in anyamplification or resulted only in a smear. The trinucleotiderepeat primers on the other hand were most effective and

Table 2. The SSR sequences, minisatellite core sequences and the arbitrarysequence decamers used as primers in amplification reactions.

S.No. Primer Name Sequence (5'-3'), Length

1 TATC-6 (TATC)6, 24 mer2 TGTC-4 (TGTC)4TGT, 19 mer3 GATA-6 (GATA)6G, 25 mer4 GACA-4 (GACA)4, 16 mer5 GTG-5 (GTG)5G, 16 mer6 CAC-6 (CAC)6C, 19 mer7 CTT-5 (CTT)5C, 16 mer8 GAA-G (GAA)6G, 19 mer9 CAC-5 (CAC)5, 15 mer10 GAA-6 (GAA)6, 18 mer11 TG-10 (TG)10, 20 mer12 AT-10 (AT)10, 20 mer13 AAAT-4 (AAAT)4, 16 mer14 AATT-4 (AATT)4, 16 mer15. 33.6a GGAGGTTTTCA, 11 mer16. HBV(5)b GGTGTAGAGAGAGGGGT, 17 mer17. HVR(-)c CCTCCTCCCTCCT, 13 mer18. OP-F02 GAGGATCCCT, 10 mer19. OP-F03 CCTGATCACC, 10 mer20. OP-U10 ACCTCGGCAC, 10 mer21. OP-U20 ACAGCCCCCA, 10 mer

aPrimer synthesized from Core sequence of 33.6 minisatellite (Jeffreys etal., 1985)bPrimer synthesized from core sequence of HBV minisatellite (Nakamuraet al., 1987)cPrimer synthesized from core sequence of HVR minisatellite (Winberg etal., 1983)

Table 3. Determination of the optimum annealing temperature for thedifferent microsatellite or SSR primers. Td was calculated as described inthe materials and methods. The shaded areas indicate that the best profileswere obtained at that temperature for the primer. The PCR results areindicated as P for multibanded profiles, NP for no profiles and S for smearsor fuzzy bands.

S. No. Primer Name Td (C) Annealing temperature (C) as in Table 2 and Result of PCR

1 TATC-6 60 51, NP 54, NP 57, P2 TGTC-4 56 45, NP 48, NP 52, NP3 GATA-6 64 54, NP 57, NP 61, S4 GACA-4 48 38, P 42, P 45, P5 GTG-5 54 45, P 49, P 51, P6 CAC-6 64 54, P 57, P 61, P7 CTT-5 44 34, NP 38, NP 42, NP8 GAA-G 52 42, P 45, P 47, P9 CAC-5 50 42, S 45, S+P 47, S10 GAA-6 48 38, P 42, P 45, P11 TG-10 60 51, NP 54, NP 57, S12 AT-10 40 30, NP 34, NP 38, NP13 AAAT-4 32 22, NP 26, NP 30, NP14 AATT-4 32 22, NP 26, NP 30, NP

To overcome these limitations PCR amplification wascarried out using a single primer composed of repeatsequence and this strategy was known as Single PrimerAmplification Reaction (SPAR, Gupta et al., 1994; Weisinget al., 1995). When mini- or micro-satellite sequences areindividually used as primer for amplification, they almostinvariably show extensive polymorphism due to site-specificlength variation in the inter-repeat region. As aconsequence different number of bands (markers) areproduced. In case of microsatellite primers for SPAR, theresulting pattern of amplified DNA corresponds to theregions between two successive SSRs (Inter SimpleSequence Repeat-PCR; ISSR-PCR). The SPAR approachto minisatellite analysis has been described as DirectAmplification of Minisatellite-region DNA (DAMD), whichdirects the amplification to regions rich in minisatelliterepeats by using the core sequence of minisatellites assingle primer (Heath et al., 1993; Zhou et al., 1997).

The PCR carried out with arbitrary sequence primers(RAPD), may actually be described as the genericprecursor of SPAR since in RAPD also a single primer butof short arbitrary sequence was used. It (RAPD) is apowerful technique and has been extensively employedever since two groups working independently developed itsimultaneously (Welsh and McClelland 1990, Williams etal., 1990). We have earlier used this technique to scoregenetic variation amongst 34 provenances of neem(Farooqui et al., 1998). Similarly, the technique was alsoused for detecting inter- and intra-species variationamongst Prosopis accessions and species (Goswami andRanade, 1999).

In case of neem, no prior sequence information onthe genome is available even though it is an ancient and


resulted in distinct amplification profiles with most of theprimers revealing broadly similar profiles across theprovenances.

DNA from 15 different provenances (Table 1) wasamplified using the primers according to optimumconditions as described above. In experiments with (GAA)6,(GAA)6G and (CAC)5 most informative patterns wereproduced at 45C which is the optimal annealingtemperature for these primers. Primers with repeat motifs(TGTC), (CTT), (AT), (AAAT) and (AATT) did not produceany profiles at three different annealing temperatures tested(Table 3). The profiles obtained after amplification of DNAsof 15 provenances with (GAA)6G, (CAC)5 and (GAA)6 aredepicted in Figure 1a to 1c respectively. The profilesobtained were mostly similar between provenances.However, the numbers of amplification products(corresponding to ISSR-regions) were different betweenthe provenances while there were no obvious bands of

different sizes (polymorphic) that were present. The numberof ISSR-regions amplified ranged from 3 in case of someof the provenances with (GAA)6G as primer to as many as10-14 in case of (CAC)5 and (GAA)6 as primers. In general,the products obtained were of low molecular weight, in therange of 1200-180bp with (GAA)6G primer and 2100-220bpwith (CAC)5 and (GAA)6 primers. Provenance number 12in case of (GAA)6G and provenance number 25 in case of(GAA)6 primer did not result in any profile (Figure 1a, 1crespectively). However, the same DNAs did reveal goodprofiles with the other primers tested.

SPAR with Minisatellite PrimersDAMD PCR was carried out essentially according to Zhouet al. (1997) using Idaho Air Thermal Cycler. There weretwo to ten clearly distinguishable bands in the differentsamples of neem, with distinctly polymorphic bandingpatterns, after amplification with the 33.6 minisatellite primer

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 amplificationreactions are analyzed by electrophoresis through 2% (w/v) agarose gels in TBE buffer made according to Sambrook et al., (1989). The numbers above thelanes correspond to the provenance sample numbers as given in Table 1. The lane marked with M contains DNA molecular weight marker ( DNA doubledigested 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 (Twobands 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).


(Figure 2). In the case of profiles obtained with 33.6minisatellite core sequence primer, provenance numbers24, 29 and 63-65 failed to amplify. Therefore, theseprovenances were not considered for the UPGMA analysis.Though no individual specific profile was obtained, therewere several small groups of samples with similar types ofprofile (Figure 3) even in the UPGMA dendrogram. Thoughthe eight exotic provenances (five from Thailand, two fromPakistan and one from Nepal) grouped together, these,

however, did not separate out as an outgroup. Theamplification 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 distinctsince only faint bands were visible along with backgroundsmear. These two profiles (with HBV5 and HVR primers)did not improve even after varying different reactionparameters and annealing stringency conditions (data notshown).

Figure 2. DAMD-PCR profiles of neem provenances using 33.6 minisatellite core primer. The products of the amplification reactions are analyzed byelectrophoresis through 2% (w/v) agarose gels in TBE buffer made according to Sambrook et al., (1989). The neem provenances are identified with numberscorresponding to the list in Table 1, above the lanes, while lanes marked with M contain DNA molecular weight marker ( DNA double digested with EcoRIand 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 4.97kb)

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


RAPD-PCRThe RAPD-PCR profiles in case of the 14 provenancesobtained with the four primers OP-F02, OP-F03, OP-U10and OP-U20 are given in Figure 4 a, b, c, d respectively.With primers OP-F02 and OP-F03, some of the bands wereobserved in all 14 provenances (indicated with an arrow,Figure 4a,b) indicating that these had presumably beenamplified from conserved genomic regions. Similarly withprimers OP-F02 and OP-U20 all the Thailand provenancesshowed the presence of bands that were not present inany other provenance (indicated with an arrowhead, Figure4a,d).

Discussion

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

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 correspondto 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).


(Langercrantz et al., 1993). Due to their varying repeatnumbers at a given locus, the elements frequently changetheir length by slipped strand mispairing, the surroundingsingle copy sequence are normally not affected andtherefore provide a valuable source of polymorphism foridentification of species and cultivars (Tsumura et al., 1996;Fang and Roose, 1997). These have been used to assessgenetic diversity even in inbred lines of dent corn andpopcorn (Kantety et al., 1995). ISSR polymorphic DNA wasevaluated for its applicability as a genetic marker systemand compared with the other commonly used markers asRFLPs and RAPDs by Nagaoka and Ogihara (1997), incase of wheat. The genetic relationships of wheataccessions estimated by ISSR markers were identical tothose inferred by RFLP and RAPD markers, indicating thereliability of ISSR markers for the estimation of genotypes.The potential of ISSR-PCR for fingerprinting purpose wasrecently evaluated in case of potato where only fourmicrosatellite primers were found to be sufficient forcomplete diagnosis of all the cultivars (Prevost andWilkinson, 1999).

In the present study, the SSR primers were annealedat different temperatures that were between 2C to 10Clower than the Td of the primer, so as to determine theoptimum temperature for a particular microsatellite. Of the14 primers tested, only 7 resulted in patterns. This is similarto the situation in case of Citrus ISSR markers whereinout of 46 primers screened, only 22 resulted in profileswhile the rest either did not produce any profiles or resultedin a smear of fuzzy band patterns (Fang and Roose 1997).The trinuleotide repeat primers were most effective andresulted in amplification profiles with most of the primersrevealing broadly similar profiles across the provenances.The patterns were reproducible among experiments, DNAsamples and primer lots. Most of the tetranucleotide repeatprimers on the other hand were ineffective and showedsmears or fuzzy and weak band profiles. Such profilesreflect either the abundance of these repeats in the genomeor that such patterns may be the result of amplificationdue to annealing to complementary strands of the repeatto each other to form concatamers (Jeffreys et al., 1988).Surprisingly none of the AT rich primers resulted in anyprofile. One possible reason for this could be due to lackof AT rich repeats in the genome of neem. If this were true,neem would apparently contrast with the reported trend ofabundance of (AT)n sequences in plants (Condit andHubbell, 1991; Akagi et al.,1997).

The profiles obtained with SSR primers consisted ofseveral closely spaced bands and no fragments that wereobviously polymorphic amongst the genotype could beidentified. This was surprising since these results indicatedthat the ISSR regions were also apparently conserved atleast in length if not in sequence. This is apparently truesince the neem template DNAs pre-digested with selectedrestriction endonucleases prior to use for PCR with(GAA)6G, (GAA)6 and (CAC)5 as primers also did not revealany large-scale variations amongst the provenances (datanot shown). It was expected that the pre-digestion of theDNA with the restriction endonuclease may revealdifferences across the provenances in their ISSR regionsthat were amplified with the SSR primer, in the form ofdistinctly polymorphic bands. Furthermore, the recent

success with ISSR-PCR in case of plants like Citrus (Fangand Roose, 1997) and wheat (Prevost and Wilkinson, 1999)indicated the resolution power of this technique andtherefore, we expected large-scale polymorphism to havebeen revealed even amongst the small group ofprovenances that we selected. These provenances in mostcases are hundreds of kilometers apart. Our results indicatethat inspite of this, ISSR regions of these provenances aremostly similar in length and possibly in sequence too. Thiscan only be possible if the provenances share a greatergenetic relatedness.

DAMD-PCRHeath et al., (1993) developed a novel technique called asDAMD, which uses PCR to direct the amplification toregions rich in minisatellites. In DAMD-PCR, a single primerfrom a minisatellite core is used to direct PCR from theregions rich in minisatellites. These regions may havesequences involved in inversions between successiveminisatellites resulting in their distribution on both strandsin opposite orientations. It is to these regions that the coreprimer anneals and results in a discrete profile of the inter-successive-minisatellite region. If inversions have takenplace, then the repeats would most likely have a piece ofsingle copy flanking DNA inserted between them.Therefore, DAMD could also amplify sequences formerlyadjacent to hypervariable minisatellite loci. Furthermore,since minisatellite core sequences are conserved acrossspecies and are larger than RAPD primers, DAMD-PCRcan be effectively carried out at relatively high stringencies,thus yielding highly reproducible results (Heath et al., 1993).

By means of highly specific M13-PCR fingerprintingtechnique profiles were determined in case of Willows(Chong et al., 1995) while the mutant and revertant tissueswere analyzed in case of Quercus sp. by Fladung andZiegenhagen (1998). M13 primers have also been used todifferentiate between 32 megagametophytes, which wereinvestigated from seeds of a single silver fir (Abies alba)tree (Degen et al., 1995). PCR-primed with wheatminisatellite core sequences yielded DNA-fingerprintingprobes in wheat (Bebeli et al., 1997). Hence the minisatellitecore primers are suggested to be universally applicable inPCR-fingerprinting experiments enabling the geneticdifferentiation of individuals.

Distinctly polymorphic banding pattern was observedwhen minisatellite core sequence primer 33.6 was usedwith neem DNA as template. Since the PCR reactions weredone at high stringency, DAMD-PCR yielded highlyreproducible results with all samples tested. However, noindividual specific profiles were obtained. Few groups ofprovenances with similar patterns were produced, showingsimilar kind of genome organization. These observationssuggested that DAMD-PCR, successfully amplified theneem genomic DNA, producing RAPD like results for theidentification of specific region in the genome. The othertwo primers HVR (-) and HBV5, did not reveal any distinctprofiles. This could be due to absence or less occurrenceof an inversion site of minisatellite region or in general alow homology of these core sequences to neemminisatellite families unlike in case of rice where these haveresulted in discrete profiles (Zhou et al., 1997). The samplesfor DAMD-PCR study included provenances from Nepal,


Pakistan and Thailand also. However, even these exoticprovenances did not reveal any appreciable differencesrelative to the Indian neem provenances, as far as theirinter-minisatellite regions are concerned.

RAPD-PCRThe RAPD studies, whenever, carried out for inter-speciescomparison, invariably resulted in clear distinction ofspecies enabling phylogenetic and taxonomicinterpretations (Adams and Demeke 1993; Abo-Elwafa etal., 1995; Millan et al., 1996). Thus it was expected thatthe Thailand provenances (considered to represent adifferent species A.siamensis) should be clearlydistinguished from all other provenances that representthe species A.indica. Though in our study with RAPDprimers, we have observed amplification products specificto Thailand neem provenances in case of a few of theprimers (Figure 4a,d), this trend was not consistent for allthe primers tested. However, Singh et al. (1999) have usedAFLP to clearly distinguish between Indian and Thailandneem provenances on the basis of a large number of AFLPmarkers. The clear distinction of Thailand provenancesfrom all others by AFLP technique but not by the RAPD orSPAR techniques indicates that a larger number of markersare required for this. This suggests that the two groups ofprovenances are more similar than dissimilar. Thus eventheir ISSR or inter-minisatellite regions are not so welldifferentiated that the Thailand neem provenancesseparate out in the dendrogram as outgroups.

The results of this SPAR study with SSR, minisatelliteand RAPD primers, carried out for the first time to ourknowledge in case of neem has thus indicated the existenceof a greater-than-expected genetic similarity amongst neemprovenances. These results agree well with our earlierassessment of a narrow genetic base in case of neem usingonly the RAPD profiles (Farooqui et al., 1998). Thus neemappears to have spread in India starting from only a fewgroups of plants. It is also possible that neem thoughcurrently widely distributed in India, did not originate here.If this is indeed so, neem in India must have evolved fromthe original founder neem trees introduced long ago, andthis may well explain the lack of obvious genetic diversityamongst the provenances selected for the present study.

Experimental Procedures

Plant MaterialThe neem provenances selected for the present study withthree types of SPAR, namely, ISSR-PCR, DAMD-PCR andRAPD-PCR, are listed in Table 1. For ISSR-PCR, weselected 15 provenances from geographically separatedregions. For the DAMD-PCR, however, we selected a widerrange and larger number of provenances since weassumed that the minisatellites having longer sequencemotifs than the SSRs might not reveal any large-scalevariation. For the RAPD-PCR, we selected 14 provenancesthat included five from Thailand, two from Pakistan as exoticprovenances, and three from Lucknow, four from Kanpuras Indian provenances.

SSR, Minisatellite and RAPD PrimersFourteen SSR primers were custom synthesized fromRama Biotech (Secunderabad, India) and CBT (New Delhi,India), while the core sequences of three minisatellites werecustom synthesized from Bangalore Genei, Bangalore,India. The four RAPD primers were procured from OperonTech. Inc., Alameda, CA, USA. The sequences of all theseprimers are given in Table 2.

DNA IsolationThe total leaf tissue DNAs from the neem plants fromvarious provenances were isolated as described earlier(Farooqui et al., 1998). The samples were checked bothqualitatively as well as quantitatively by agarose gelelctrophoresis according to Sambrook et al. (1989).

SPAR with SSR PrimersThe PCR conditions as proposed by Gupta et al. (1994)were used with minor modifications in concentration ofmagnesium ions. A pilot experiment was carried out todetermine the optimum concentration. The final reactionwas carried out in 10 l volumes and contained 50 ng oftemplate DNA (quantitation was done by DynaQuant200fluorometer), 10 pmoles of SSR primer, 200 M each dNTP,3.0 mM Mg2+ ion concentration in suitable 1X assay buffersupplied along with the enzyme and 0.5 Units of thethermostable Taq DNA polymerase (Bangalore Genei,Bangalore, India). The tubes were placed in the Air ThermalCycler (Idaho Technology, USA) for the PCR. The AirThermal Cycler was programmed to include pre-denaturation at 94C for 1 minute. This was followed by25 cycles of denaturation at 94C for 30 s, annealing atthe optimized temperature for 30 s, and extension at 72Cfor 1 min. The final cycle allowed an additional 5 min periodof extension at 72C. When all the reaction componentswere standardized the annealing temperature wasoptimized for each SSR primer. The optimum annealingtemperature was determined in the range of 2-10C lowerthan the denaturation temperature (Td) as shown in Table3. The denaturation temperature was calculated accordingto Berger and Kimmel (1987), by adding 2C for each A orT and 4C for each G or C in the oligomer. The reactionproducts obtained after PCR were analyzed on 2% agarosegel. The gel was stained in ethidium bromide and visualizedon Nighthawk gel documentation system (pdi Inc., USA).Only distinct and well-separated bands were consideredfor the analysis.

SPAR with Minisatellite PrimersThe reaction components were similar to that as optimizedfor SSR primers. The reaction mixtures contained 50 ng oftemplate DNA, 10 pmoles primer, 3.0 mM magnesium ions,200 M each dNTP, 0.5 Units enzyme and 1X buffer. Thevolume was made upto 10l with deionized sterile water.The DAMD-PCR was carried out essentially according toZhou et al. (1997). The optimum annealing temperatureswere determined by carrying out PCR at three annealingtemperatures 47C, 50C and 55C. Of the threetemperatures, 55C annealing temperature resulted in theclearest profile. The cycling parameters were as follows:First cycle of 94C for 1 minute for initial denaturation of


template, then 35 cycles of incubation at 94C for 30seconds, at annealing temperature (55C) for 1 minute and72C for 1 minute. Last cycle allowed 5 minute at 72C forextension. These conditions were used with all minisatelliteprimers. The products obtained were electrophoresed on2% agarose gel in 0.5X TBE buffer at 5V/cm. Afterelectrophoresis the gel was stained in ethidium bromideand then visualized and photographed on geldocumentation system. Only distinct and well-separatedbands were included in the analysis.

RAPD of Neem ProvenancesFor the RAPD studies, a smaller set of 14 provenanceswas selected from amongst the 52 used for DAMD-PCRand ISSR-PCR. While selecting this set, we have includedmultiple provenances from a given region. RAPD analysiswas 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 andcontained 50 ng of template DNA, 10 pmoles of arbitrarysequence decamer primer, 200 M each dNTP, 2.5 mMMg2+ ion concentration in suitable 1X assay buffer suppliedalong with the enzyme and 0.5 Units of the thermostableTaq DNA polymerase (Bangalore Genei, Bangalore, India).The tubes were placed in the Air Thermal Cycler (IdahoTechnology, USA) for the PCR. The Air Thermal Cyclerwas programmed to include pre-denaturation at 94C for1 minute. This was followed by 45 cycles of denaturationat 94C for 30 s, annealing at 35C for 30 s, and extensionat 72C for 1 min. The final cycle allowed an additional 5min period of extension at 72C. The PCR products wereanalyzed by electrophoresis through 1.0 % agarose gelsas above.

Acknowledgements

We thank the Biomass Research Center and Arid ForerstResearch Institute at Lucknow and Jodhpur respectively,for all help with provenance selection. Financial supportas Senior Research Fellowship from the CSIR to NF andgrant-in-aid from DBT to SAR are gratefully acknowledged.We are thankful for support and encouragement of Director,NBRI.

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Assessment of Profile Variations Neem SPAR

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