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Use of RAPD markers to detect sex differences in Pandanus tectorius Parkinson, an important bioresource plant in Orissa, India

Kamal K. Pandaa; Biswajit Sahooa; Anath B. Dasb; Brahma B. Pandaa a Berhampur University, Molecular Biology and Genomics Laboratory, Department of Botany, Berhampur, India b Cytogenetics Laboratory, Regional Plant Resource Centre, Bhubaneswar, India First published on: 05 July 2010

To cite this Article Panda, Kamal K. , Sahoo, Biswajit , Das, Anath B. and Panda, Brahma B.(2010) 'Use of RAPD markers

to detect sex differences in Pandanus tectorius Parkinson, an important bioresource plant in Orissa, India', International Journal of Biodiversity Science, Ecosystem Services & Management, 6: 1, 28 — 34, First published on: 05 July 2010 (iFirst) To link to this Article: DOI: 10.1080/17451590.2010.487472 URL: http://dx.doi.org/10.1080/17451590.2010.487472

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International Journal of Biodiversity Science, Ecosystem Services & Management Vol. 6, Nos. 1–2, March–June 2010, 28–34

Use of RAPD markers to detect sex differences in Pandanus tectorius Parkinson, an important bioresource plant in Orissa, India Kamal K. Pandaa, Biswajit Sahooa, Anath B. Dasb† and Brahma B. Pandaa*

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a Molecular Biology and Genomics Laboratory, Department of Botany, Berhampur University, Berhampur 760 007, India; bCytogenetics Laboratory, Regional Plant Resource Centre, Bhubaneswar 751015, India

Pandanus tectorius Parkinson (¼ P. fascicularis Lam.) is a dominant perennial dioecious plant of coastal areas of India. Aromatic inflorescences harvested from male plants constitute the raw material that supports a flourishing perfume industry confined to the Ganjam coast on the east coast of south Orissa. The plant is vegetatively propagated, and sex is only clear after 5–7 years when plants from stem cuttings start to flower. Early determination of the sex of the plant was examined through analysis of somatic chromosome compliment, genomic DNA content and random amplified polymorphic DNA (RAPD) analysis in seven populations of P. tectorius from the coast of Orissa. Whereas the chromosome complement (2n ¼ 60) failed to reveal any differences, the 4C DNA amount indicated that the genome of female plants [6.15 pg ¼ 5935 mega base pair (Mbp)] was significantly larger than that of male plants (5.09 pg ¼ 4912 Mbp). Motivated by the perfume economy, local farmers presently look upon P. tectorius as a cash crop and tend to conserve and propagate male genotypes of several populations along the coast of Ganjam. The two RAPD markers, 1150 and 600 bp, amplified with primers OPB-12 and OPN-18, respectively, may together be useful to conserve elite genotypes in natural populations of P. tectorius. Keywords: bioresources; essential oils; Pandanaceae; random amplified polymorphic DNA (RAPD)

Introduction Pandanus tectorius Parkinson (¼ P. fascicularis Lam.), locally called ‘Kewda’ or ‘Kia’, belongs to the Pandanaceae and is a wild and dominant perennial species in the coastal vegetation of Orissa (Panda et al. 2009). Traditionally, the plant was used for fencing agricultural fields to protect them from cattle grazing. The plant grows profusely near the seashore in xeric conditions, mainly along the coast, providing ecological benefits such as arresting soil erosion and forming the basis of sand dunes, thus acting as a natural bulwark against wind. This plant is dioecious, with distinct male and female inflorescences borne on different plants. The male inflorescence constitutes the raw material from which the aroma ‘rhu’ or essential oil (phenyl ethyl methyl ether, terpine-4-ol, p-cymene, a-terpineol, etc.) is extracted (Panda et al. 2007, 2009). Male plants have been supporting the flourishing perfume industry that is presently confined to Ganjam District along the coast of Orissa. Generally, female inflorescences (and fruits) are of little use. Thus the plant is an important coastal bioresource that constitutes a key component of the local agroecosystem, contributing significantly to the socio-economy of the region (Panda et al. 2007, 2009). Evolution and differentiation of distinct male and female plants (dioecism) promote natural hybridisation through cross-pollination. Sex chromosomes showing the XY system are known in several dioecious plants, in which males are heterogametic and females are homogametic (Dellaporta and Calderon-Urrea 1993; Matsunaga and Kawano 2001). The

genome constitutes the haploid DNA content (C) of an organism. Feulgen cytophotometry of mitotic chromosome complement estimates 4C DNA. In situ estimation of 4C DNA provides clues pertaining to sex differences leading to dioecism (Bennett and Leitch 1995). DNA markers, which are not prone to environmental influence, can accurately identify sex in dioecious plants, thereby overcoming the need to wait till flowering for sex determination. There are many methods to determine sex of a plant, including restriction fragment length polymorphisms (RFLP) e.g. in Asparagus (Biffi et al. 1995); sex-linked amplified fragment length polymorphism (AFLP), e.g. in Poa arachnifera (Renganayaki et al. 2005); microsatellite (GATA)n-based banding patterns, e.g. in Carica papaya (Parasnis et al. 1999) and random amplified polymorphic DNA (RAPD), with or without sequence characterized amplified regions (SCAR), which has been used extensively, e.g. in Salix viminalis (Alstrom-Rapaport et al. 1998), Asparagus (Jiang and Sink 1997), Piper longum (Banerjee et al. 1999), Cannabis sativa (Mandolino et al. 1999), Salix viminalis (Gunter et al. 2003), Eucommia ulmoides (Xu et al. 2004), Simmondsia chinensis (Agrawal et al. 2007), Carica papaya (Chaves-Bedoya and Nunenz 2007) and Pandanus fascicularis (Vinod et al. 2007). Seed production through sexual means, as well as propagation through seeds, is unknown in P. tectorius. The plant is propagated from vegetative stem cuttings, and sex is determined only when these plants flower, normally after 5–7 years. The male inflorescences, from which essential oil is

*Corresponding author. Email: [email protected] † Present address: Molecular Cytogenetics Laboratory, Department of Agricultural Biotechnology, College of Agriculture, Orissa University of Agriculture and Technology, Bhubaneswar 751003, India. ISSN 2151-3732 print/ISSN 2151-3740 online # 2010 Taylor & Francis DOI: 10.1080/17451590.2010.487472 http://www.informaworld.com

International Journal of Biodiversity Science, Ecosystem Services & Management distilled, constitute the raw material for the perfume industry. In order to augment commercial production of male inflorescences to support the local perfume industry, early identification of the male genotypes of P. tectorius is desirable for their large-scale vegetative propagation. The present paper uses analyses of the somatic chromosome compliment, 4C DNA content and RAPD of male and female plants from seven populations (I–VII) obtained from the coast of Orissa.

Materials and methods

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Study sites A survey along the coast of Orissa and bordering Andhra Pradesh was undertaken covering a 600 km from Palasa to Balasore, through Ganjam, Puri and Paradeep. Stem cuttings from eight different male and female plants of P. tectorius from seven locations (populations I–VII) were sampled, from a grid with random of intersection points from the coast of Orissa (Figure 1). Stem cuttings were subsequently transplanted and maintained in earthen pots in the Botanical Garden, Berhampur University, Berhampur. Percentage success of rooting of cuttings following transplantation was assessed. Eco-geographical attributes such as latitude/longitude, pH and soil type with respect to each location are presented in Table 1. Each population consisted of ,1000 plants each covering an area of 10 ha. The male to female ratio in each population was about 20:1, and age of the populations was .20 years.

29

Cytological analysis Root tips from young roots from each cutting were excised, washed thoroughly in water and pre-treated with 0.002 M aqueous hydro-oxyquinonine for 4 h at 25 C, followed by overnight fixation in acetic acid:ethanol (1:3 v/v). Metaphase chromosome spreads were prepared following squashing of haematoxylin-stained root tips as described earlier (Panda et al. 2007). Thirty well-separated metaphase plates were examined at 1000 under an oil-immersion microscope from 10 slides prepared from 10 root tips obtained from five different male or female cuttings per population, and representative chromosome spreads were photographed and recorded from an Olympus 51BX microscope with a Cohu 4910 monochrome high-resolution CCD video camera.

Feulgen cytophotometric estimation of 4C DNA content For Feulgen cytophotometric estimation of 4C DNA content, 10 root tips from each of five different plants per population (male and female) were fixed in acetic acid:ethanol (1:3) and hydrolysed in 1 N HCl at 60 C for 15 min; washed in distilled water and rinsed in SO2 water (Fox 1969). Roots were then stained in Feulgen reagent for 2 h in the dark and root tip squashes were made on a slide in a drop of 45% acetic acid. The 4C DNA content was estimated from at least 10 metaphase plates per slide and 10 slides using a Nikon Optiphot microscope fitted with a micro-spectrophotometer with a 550 nm filter (Sharma AK and Sharma A 1980). DNA values

Figure 1. Map showing the sampling sites: I. Palasa, II. Tulu/Indrakhi, III. Chatrapur, IV. Chilika,V. Puri/Konark, VI. Paradeep/Ersama and VII. Balasore along the coastline of Orissa from where the male or female plants of P. tectorius were obtained for investigation in the present study.

30 Table 1.

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Sl. no.

K.K. Panda et al. Location and physical characteristics along the coast of Orissa of populations I–VII of P. tectorius investigated. Populations

District/state

I

Palasa

Srikakulaum (Andhra Pradesh)

II

Tulu/Indrakhi

Ganjam (Orissa)

III

Chatrapur

Ganjam (Orissa)

IV

Chilika

Khorda (Orissa)

V

Puri/Konark

Puri (Orissa)

VI

Paradeep/Erasma

Jagatsinghpur (Orissa)

VII

Balasore (Orissa)

Balasore

obtained on the basis of optical density of the metaphase chromosome were converted to picograms (pg) using the 4C nuclear DNA value (67.1 pg) for Allium cepa L. var. Deshi (Van’t Hof 1965) as standard. Furthermore, 4C DNA amount was expressed in megabase pairs of nucleotides (1 pg ¼ 965 Mbp; Bennett and Leitch 1995).

DNA extraction and purification Young, fresh leaf tissue from eight male or female plants per population was used for genomic DNA following the standard CTAB protocol (Saghai-Maroof et al. 1984) with some modifications. Bulk (10 g) samples were prepared from at least 1 g of tissue per sample, ground in liquid nitrogen and suspended in 20 ml CTAB extraction buffer [100 mM Tris HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2.5% cetyl trimethyl ammonium bromide (CTAB), 2% b-mercaptoethanol]. The suspension was incubated at 55 C for 2 h, extracted with an equal volume of chloroform:isoamyl alcohol (24:1) and centrifuged at 10,000 g for 15 min. The aqueous phase was transferred to a new 50-ml tube and DNA precipitated with two-thirds volume of chilled isopropanol. The DNA was spooled out and dissolved in T10E1 (10 mM Tris-HCl, 1 mM Na2EDTA, pH 8.0), then treated with RNAseA (10 mg ml1, MBI Fermantas, Vilnius, Lithuania) at 37 C for 1 h. The DNA was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), centrifuged at 10,000 g for 15 min, the aqueous layer was transferred to a fresh tube and re-extracted with an equal volume of chloroform:isoamyl alcohol (24:1) by centrifuging at 10,000 g for 15 min. The supernatant was transferred to a fresh tube and precipitated in chilled ethanol with 3 M sodium acetate buffer pH 5.2. DNA was spooled out, washed in 70% ethanol, air-dried, dissolved in T10E1 buffer and kept in a deep freeze at 20 C. The concentration of DNA was estimated at 260 nm using a UV/Vis spectrophotometer (ECIL, Hydrabad, AP, India). Subsequently, purity and quality of DNA was assessed at A260/A280 ratio (1.8 and 2.0) as well as following electrophoresis on 0.8% agarose gel. The DNA was diluted to a final concentration of 25 ng ml1 with T10E1 buffer for use as template in polymerase chain reaction (PCR) analysis.

Latitude/longitude 18 900 N 84 780 E 19 180 N 84 550 E 19 280 N 85 020 E 19 400 N 85 270 E 19 840 E 85 850 N 20 170 N 86 350 E 21 260 N 86 530 E

Salinity (ppt)

Soil pH

Soil type

10–22

7.2–9.6

Clay/sandy

12–19

6.5–7.8

Clay

12–16

6.8–7.6

Alluvial

11–27

6.8–9.7

Fine sand

14–18

6.5–8.82

Sandy loam

9–17

5.9–7.2

Fine silt clay

7–14

5.8–7.3

Clay/fine sand

PCR amplification and RAPD analysis RAPD profiles were generated using single decamer primers (Operon Technologies, Alameda, CA, USA) and PCR following Williams et al. (1990). Each amplification reaction mixture contained 25 ng template DNA, 200 mM dNTP mix (MBI Fermantas), 25 ng primer, 1 unit Taq DNA Polymerase (Bangalore Genei, Bangalore, India) and 10  PCR assay buffer (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, pH 9.0) in a final volume of 25 ml. The reaction was carried out in a thermal cycler (BioRad, Benicia, CA, USA). The first cycle consisted of denaturation at 94 C for 3 min, followed by 45 cycles of 1 min denaturation at 94 C, 1 min primer annealing at 38 C and 2 min DNA polymerisation at 72 C, followed by primer extension (72 C) for 15 min. The amplified products were stored at 4 C and separated electrophoretically on 1.5% agarose gel in 1 TAE buffer under constant voltage (55 V) for 4 h. A low range DNA ladder (100–3000 bp; Bangalore Genei, India) was used as standard to size amplified RAPD bands. Ethidium bromide-stained amplified DNA bands were visualized using an UV trans-illuminator, and the image captured with a Nikon Coolpix 4S camera mounted on a hood (Biotech R&D, Yercaud, India), and subsequently transferred to a computer. DNA amplifications (amplicons) generated with 12 primers were scored and used for further RAPD analysis.

Statistical analysis Mean values of total genomic 4C DNA was determined for male and female plants of different populations. ANOVA was performed among nuclear DNA values with Duncan’s multiple range test (Harter 1960). A binary matrix from the RAPD data was obtained from presence or absence of bands, but differences in their intensity were ignored. The binary matrix was transformed into a similarity matrix using Jaccard’s coefficient. From this, a phylogenetic dendrogram was obtained with cluster analysis of unweighted pairs with the arithmetic mean (UPGAM) method (Sneath and Sokal 1973). The entire analysis was performed with the statistical package NTSYS version 2.02e (Rohlf 1997). The RAPD

International Journal of Biodiversity Science, Ecosystem Services & Management data were further subjected to analysis of molecular variance (AMOVA) as described by Excoffier et al. (1992), using three hierarchical levels: individual, population and their regions with the GenALEX software (Peakall and Smouse 2001), and also used for principal coordinate analysis (PCA) of the relationship between the distance matrix and elements based on the first two principal coordinates.

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Results Somatic metaphase analysis revealed a chromosome number of 2n ¼ 60, with occasional variation between 2n ¼ 56 and 62, for both male and female plants, irrespective of population (Figure 2). The cytophotometric 4C DNA content of male and female P. tectorius varied significantly among male and female plants. The 4C DNA content in male plants was 4.60–5.65 pg, while female plants ranged from 5.9 to 6.31 pg. No significant intra- or inter-population differences in 4C DNA content were noted among male and female plants; however, the difference between average male (5.09 pg % 4912 Mbp) and female (6.15 pg % 5935 Mbp) 4C DNA was highly significant (p  0.01). Since in pilot experiments RAPD analysis did not reveal any significant intra-population differences among male or female plants, purified genomic DNA isolated separately from bulk leaf tissue of male or female plants for each population was analysed. Out of 30 primers of the OPA, OPC, OPD and OPN series tried initially for DNA amplification, 12 generated consistent and reproducible amplicons in three or more repeat experiments (Table 2). The size of amplicons ranged from 300 to 1800 bp in all populations. A total of 798 bands were amplified, out of which 404 were in male and 394 were in female populations. Of the tested primers, only two primers, OPA-12 and OPN-18, showed high polymorphism, from which, irrespective of population, male plants could be distinguished from female plants. A marker band of 1150 bp was produced by OPA-12 in all male plants in all populations. OPN-18 amplified a distinct marker band of 600 bp, which was characteristically associated with all male plants from four populations, I–IV (Figure 3)

Figure 2.

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confined to the Ganjam coast and immediate neighbourhood. Remarkably, none of the aforementioned male sex-linked marker bands was associated with female plants of any population investigated. The matrix of the similarity index (Table 3) and resulting dendrogram (Figure 4) unambiguously segregated two distinct clusters of male or female plants. Furthermore, the male plants formed two sub-clusters, one with populations I–IV and the other with populations V–VII, both showing relatively more genetic homogeneity than their female counterparts. Female plants formed two sub-clusters, comprising populations I, III, VI and VII in sub-cluster I and populations II, IV and V in sub-cluster II. Furthermore, within sub-cluster I, population I was distantly related to populations III, VI and VII, out of which populations VI and VII were genetically homogeneous. In subcluster II, population V was separate from populations VI and II, indicating genetic divergence; the latter two with high homogeneity are placed together. AMOVA helped to partition the RAPD variations among sexual groups and among populations within a group. About 3% of molecular variance was among populations, while within populations it was ,95%, indicating higher variation between male and female populations. This may be useful in strategies for germplasm collection and evaluation. The PCA analysis (Figure 5) was comparable to the cluster analysis (Figure 4), with all male genotypes distinctly different from female genotypes. Male genotypes from Palasa (I), Tulu (II), Chatrapur (III) and Chilika (IV) that represent the Ganjam coast and its immediate neighbourhood were distinct from othermale genotypes in the PCA. However, all female genotypes from all populations formed a separate group.

Discussion The somatic chromosome number was 2n ¼ 60 in both male and female populations of P. tectorius, with occasional mosaicism, which is quite common in vegetatively propagated plants (Panda et al. 2007). Although there was no marked difference in chromosome number between male

Metaphase spreads of P. tectorius, showing 2n ¼ 60 in male (A) and female (B). Bar indicates 10 m.

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Table 2. RAPD primers and number and size of RAPD bands generated from seven male and female populations (I–VII) of P. tectorius from the coast of Orissa. Sl. no.

OPA 10 OPA 11 OPA 12 OPA 13 OPA 14 OPC 07 OPC 19 OPD 09 OPD 15 OPN 09 OPN 14 OPN 18

Primer sequence (nucleotide bases) 0

Total no. of bands

Male plants

Female plants

Size range (base pairs)

28 73 35 63 31 86 85 56 112 56 127 46

14 36 21 35 14 42 42 28 56 28 63 25

14 37 14 28 17 44 43 28 56 28 64 21

500–700 600–1300 500–1200 600–1000 500–800 800–1800 800–2000 600–1500 500–1800 300–1000 500–1800 400–1000

0

5 GTGATCGCAG3 50 CAATCGCCGT30 50 TCGGCGATAG30 50 CAGCACCCAC30 50 TCTGTGCTGG30 50 GTCCCGACGA30 50 GTTGCCAGCC30 50 CTCTGGAGAC30 50 CATCCGTGCT30 50 TGCCGGCTTG30 50 TCGTGCGGGT30 50 GGTGAGGTCA30

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1 2 3 4 5 6 7 8 9 10 11 12

Primer

Figure 3. Comparative RAPD profiles of male (M) and female (F) populations I–VII of P. tectorius along the coastline of Orissa, showing male specific markers 1150 and 600 bp amplified by OPA-12 (A) and OPN-18 (B), respectively. L indicates DNA ladder in base pairs (bp).

and female plants, the genome size of female plants (6.15 pg, 5935 Mbp) was significantly higher than that of male plants (5.09 pg, 4912 Mbp) for 4C DNA content. The small chromosome size prevented determination of chromosome volume, therefore we could not correlate this to 4C DNA amount. Extraction of good quality DNA is fundamental for satisfactory RAPD analysis. Presence of excess secondary metabolites in aromatic plants is often a serious problem for DNA extraction (Padmalatha and Prasad 2006). This was overcome with minor modification to the DNA extraction protocol, with a 2-h incubation of leaf homogenate at 55 C in

chloroform:isoamyl alcohol (24:1). Furthermore, following RNAse treatment DNA was extracted again with phenol:chloroform:isoamyl alcohol (25:24:1). RAPD techniques have been widely employed for identification of sex in dioecious plants. For example, a RAPD band from UBC of 354–560 bp was shown to be linked to a sex determination locus in Salix viminalis (Alstrom-Rapaport et al. 1998); two RAPD bands, 757 bp amplified with OPC12 and 908 bp with OPA-10, were associated with male Piper longum (Banerjee et al. 1999); using 158 RAPD primers, a male-specific 2075-bp band was identified in Atriplex garretti (Ruas et al. 1998); and a single 567-bp RAPD female

International Journal of Biodiversity Science, Ecosystem Services & Management Similarity index for male (M) and female (F) populations (I–VII) of P. tectorius on the basis of RAPD analysis.

Population

MI

FI

M II

F II

M III

F III

M IV

F IV

MV

FV

M VI

F VI

M VII

FI M II F II M III F III M IV F IV MV FV M VI F VI M VII F VII

0.95 1 0.94 1 0.92 1 0.92 0.98 0.91 0.98 0.94 0.98 0.97

0.95 0.98 0.95 0.97 0.95 0.97 0.97 0.95 0.97 0.98 0.97 0.98

0.94 1 0.92 1 0.92 0.98 0.91 0.98 0.94 0.98 0.94

0.94 0.95 0.94 0.98 0.95 0.97 0.95 0.97 0.95 0.97

0.92 1 0.92 0.98 0.91 0.98 0.94 0.98 0.94

0.92 0.97 0.94 0.95 0.94 0.98 0.94 0.98

0.92 0.98 0.91 0.98 0.94 0.98 0.94

0.94 0.98 0.94 0.98 0.94 0.98

0.92 1 0.95 1 0.97

0.92 0.97 0.92 0.97

0.95 1 0.95

0.95 1

0.95

Male I Male II Male III Male IV Male V Male VI Male VII Female I Female III

Sub-cluster-I

Cluster-I

Sub-cluster-II

Sub-cluster-I

Female VI Female VII Female II Female IV Female V

Cluster-II Sub-cluster-II

0.93

0.95

0.97 Coefficient

0.98

1.00

Figure 4. Dendrogram showing genetic divergence and affinity among male and female populations I–VII of P. tectorius along the coastline of Orissa as revealed by RAPD analysis.

0.30 Male plants M4 M1 M2 M3 High perfume quality

0.15 M5 M6 M7

Dim-2

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Table 3.

33

0.00 F1 F2 F6 F7 F3 F4 F5

–0.15

Female plants

–0.30 –0.50

0.00

0.50 Dim-1

1.00

1.50

Figure 5. Two-dimensional plot of principal component analysis of seven populations (I–VII) of P. tectorius composed of male (M) and female (F) plants. The numbers 1–7 denote respective populations. Note that the male plants M1–M4 known for high perfume quality belonging to population I–IV are geographically confined to Ganjam coast or immediate neighbourhood.

sex-specific marker was identified following screening of 100 decamer primers in Trichosanthes dioica Roxb (Singh et al. 2002). In the present study, we have shown that two male-specific RAPD marker bands, 1150-bp band amplified with OPA-12 and 600-bp band amplified with OPN-18, in the genome of P. tectorius might be useful to identify male genotypes. Chromosomal loci associated with these RAPD markers, however, remain obscure. Vinod et al. (2007) screened 89 RAPD and ISSR primers and identified a 1263-bp RAPD band amplified by primer OPO-08 only in male plants of P. fascicularis, on the basis of which they developed a male-specific SCAR marker. The present RAPD analysis clearly distinguished male from female plants of P. tectorius at the vegetative stage, irrespective of population or location within Orissa. The male-specific OPA12 1150-bp RAPD band was common to all populations at seven locations (I–VII) along the coast of Orissa, while the OPN18 600-bp RAPD marker additionally identified male plants in populations I–IV from the Ganjam coast and immediate neighbourhood. Those male genotypes, especially populations II and III, belong to the so-called ‘Kewda belt’ of the Ganjam coast and are economically important because of the high perfume quality and yield of male inflorescences that support the economy and livelihood of local people (Panda et al. 2007, 2009). There was no significant genotypic variation between male genotypes of populations I–IV, which formed a single homogenous sub-cluster; therefore these should have equal potential to support the perfume industry. The malespecific RAPD markers identified here could possibly be the basis for development SCAR markers, which would be useful not only for early and rapid determination of sex but also augment selective clonal propagation of male genotypes known for perfume quality or yield. Management of bioresources through sustainable exploitation is sine qua non for conservation of biodiversity. Motivated by the perfume industry, local farmers who presently look upon P. tectorius as a cash crop conserve and propagate male genotypes of P. tectorius from populations II and III along the coast of Ganjam (Panda et al. 2009). The two RAPD markers of 1150 and 600 bp that were amplified, respectively, with primers OPB-12 and OPN-18

34

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may together be useful to conserve elite genotypes in natural populations of P. tectorius. Male genotypes in populations I and IV, which are genetically and geographically aligned with populations II and III, should be for equal quality as a resource to support the local perfume industry in this region. Nevertheless, one should not overlook or eliminate female genotypes of P. tectorius from populations even though they apparently have no economic value. Conservation of genetic biodiversity of P. tectorius is best secured when female genotypes are conserved along with male genotypes. Acknowledgements

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The authors thank the authorities of Berhampur University and the Regional Plant Resource Centre, Bhubaneswar for extending institutional support and facilities to carry out this work. This work was supported by a grant from the Department of Science and Technology, Government of India, New Delhi, under the Women Scientists Scheme, SR/WOS-A/LS-4/2003, awarded to KKP.

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