**1. Introduction**

442 Ecosystems Biodiversity

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Betelvine (*Piper betle* L., family Piperaceae) is an important, traditional and an ancient crop of India and is a shade loving, perennial evergreen climber of tropical origin. It is generally known as "Paan" in Hindi in the Indian subcontinent and by different names in the Asiatic region and is a plant of considerable antiquity. It is distributed in several countries in including India as well as other countries of Indochina region - Indonesia, Malaysia, Vietnam, Laos, Kampuchea, Thailand, Myanmar, Singapore and the Far-East (Figure 1), where its cultivation or ethnomedicinal properties are well known. In India, betelvine is widely cultivated in the states of Uttar Pradesh, Bihar, Madhya Pradesh, Northeastern India, Maharashtra, Karnataka, West Bengal, Orissa, Andhra Pradesh, Tamil Nadu, Kerala and Andamans and almost the entire production of betel leaves is consumed fresh as a masticatory.

Betelvines are dioecious and therefore, under controlled hybridization, attempts have been made to cross different landraces and in some of these experiments, viable seed set has also been reported. However, as a crop, propagation is obligately only through vegetative means. Its cultivation in northern India under sub-tropical conditions (Figure 2) has been shown to be a unique case of plant establishment under anthropogenically regulated microclimatic conditions (Kumar 1999). Cultivated betelvine is grown in traditional farming systems many of which are managed exclusively by families or communities. The betelvine growers invariably named their cultivars with local or vernacular names. These cultivated betelvines are therefore, nothing but landraces and it is this description that will be used consistently throughout the manuscript. A survey over several years indicated between 125 to 150 local cultivars (landraces) of betelvines in India.

SPAR Profiles for the Assessment of Genetic Diversity Between

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 445

planned because there were no prior reports about chromosomal basis of gender distinction in case of betelvines. A better understanding of how gender discrimination takes place in those plants where chromosomal basis of inheritance of sex is undocumented or negligible or non-existent too is important as the study also assumes significance in understanding the resource allocation and metabolism costs attributed to gender discrimination in those plants where the economic importance of plant is defined by a metabolite or a phytochemical that is recovered from a somatic tissue. Such a study in case of a dioecious plant is expected to throw light on whether or not there is a difference in diversity of the two genders and if so

whether such differences are significant in terms of the economic values of the plant.

Fig. 2. The stages during the construction of the unique man-made structures created to regulate the micro-climate for cultivation of the betelvines in the sub-Tropical parts of India. These structures are called as "bareja" and are almost exclusively made out of natural raw materials such as palm thatch and bamboo. The panels labeled A through H depict the various stages. The small black arrows in panels A through C point to the bamboo sticks that are used to support the betelvines. The white larger white arrows in panels B and C

point to the thatch walls of the bareja sides.

Fig. 1. Schematic map showing the regions of the world where betelvine is cultivated or consumed.

Currently more than 200 landraces are cultivated in India and are often named after the localities, villages or towns where they are grown. As a result of this, though the geographical distribution of the betelvines under cultivation is vast, it is possible that the genetic variation may not be so well distributed. However, no systematic study of betelvine genetic diversity had been carried out to our knowledge. Thus the assessment of genetic variation amongst the different landraces became an important objective for which, PCRbased parallel approaches were used. We have shown, in an earlier study with different landraces using RAPD method, a clear distinction between male and female betelvines or between types of landrace groups (Ranade et al. 2002; Verma et al. 2004). A further substantiation of those results as well as an in-depth analysis was carried out using four different primer sets including RAPD primers on a specific subset of landraces clearly distinguished from each other by flowering behavior as either male or female betelvines (Figure 3). On such a set of known male and female betelvines, hereafter referred to as the "gender set" we have assessed genetic diversity using four primer types revealing polymorphic profiles from discrete but widely distributed genomic regions. These primers include (i) arbitrary sequence decamer primers amplifying from several anonymous regions; (ii) SSR primers amplifying several inter-SSR regions; (iii) minisatellite core sequence primers amplifying from miniatellite rich regions and (iv) primers derived from X and Y chromosomes of a known dioecious plant with heteromorphic sex chromosomes, *Silene latifolia* that were expected to specifically amplify from genomic regions homologous to the X or Y chromosomes in case of the male and female betelvines. Such a PCR-based study was

Fig. 1. Schematic map showing the regions of the world where betelvine is cultivated or

Currently more than 200 landraces are cultivated in India and are often named after the localities, villages or towns where they are grown. As a result of this, though the geographical distribution of the betelvines under cultivation is vast, it is possible that the genetic variation may not be so well distributed. However, no systematic study of betelvine genetic diversity had been carried out to our knowledge. Thus the assessment of genetic variation amongst the different landraces became an important objective for which, PCRbased parallel approaches were used. We have shown, in an earlier study with different landraces using RAPD method, a clear distinction between male and female betelvines or between types of landrace groups (Ranade et al. 2002; Verma et al. 2004). A further substantiation of those results as well as an in-depth analysis was carried out using four different primer sets including RAPD primers on a specific subset of landraces clearly distinguished from each other by flowering behavior as either male or female betelvines (Figure 3). On such a set of known male and female betelvines, hereafter referred to as the "gender set" we have assessed genetic diversity using four primer types revealing polymorphic profiles from discrete but widely distributed genomic regions. These primers include (i) arbitrary sequence decamer primers amplifying from several anonymous regions; (ii) SSR primers amplifying several inter-SSR regions; (iii) minisatellite core sequence primers amplifying from miniatellite rich regions and (iv) primers derived from X and Y chromosomes of a known dioecious plant with heteromorphic sex chromosomes, *Silene latifolia* that were expected to specifically amplify from genomic regions homologous to the X or Y chromosomes in case of the male and female betelvines. Such a PCR-based study was

consumed.

planned because there were no prior reports about chromosomal basis of gender distinction in case of betelvines. A better understanding of how gender discrimination takes place in those plants where chromosomal basis of inheritance of sex is undocumented or negligible or non-existent too is important as the study also assumes significance in understanding the resource allocation and metabolism costs attributed to gender discrimination in those plants where the economic importance of plant is defined by a metabolite or a phytochemical that is recovered from a somatic tissue. Such a study in case of a dioecious plant is expected to throw light on whether or not there is a difference in diversity of the two genders and if so whether such differences are significant in terms of the economic values of the plant.

Fig. 2. The stages during the construction of the unique man-made structures created to regulate the micro-climate for cultivation of the betelvines in the sub-Tropical parts of India. These structures are called as "bareja" and are almost exclusively made out of natural raw materials such as palm thatch and bamboo. The panels labeled A through H depict the various stages. The small black arrows in panels A through C point to the bamboo sticks that are used to support the betelvines. The white larger white arrows in panels B and C point to the thatch walls of the bareja sides.

SPAR Profiles for the Assessment of Genetic Diversity Between

**2. Materials and methods** 

stored at -70C till further use.

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 447

*Plant material*: Betelvine landraces were collected from three of the centers of the All India Co-coordinated Research Project (AICRP) on Betelvine at Chinthalapudi (Bapatla) in Andhra Pradesh, Sirugamani (Tiruchirappalli) in Tamil Nadu and Digraj (Sangli) in Maharastra. The landraces of betelvine for which leaf tissue were collected are listed in Table 1. Young leaf tissue was harvested from the vines, washed free of dirt and dust and then quickly mopped dry on blotting sheets. The leaves were de-ribbed and powdered rapidly in liquid nitrogen, and then either the DNA isolation procedures were immediately followed or the powdered tissue was stored at -70C till further use. The leaves of, the *Piper* species outgroup (*Piper hamiltonii*) as well as non-*Piper* outgroup, Mulberry variety 'MI-0129' were collected from NBRI, Lucknow. As in the case of betelvines the young leaf tissue was harvested, washed free of dirt and dust and then quickly mopped dry on blotting sheets. Then the leaves were de-ribbed and powdered rapidly in liquid nitrogen, and then either the DNA isolation procedures were immediately followed or the powdered tissue was

*Isolation of DNA*: Total plant DNA was isolated from the frozen tissue powder according to the method of de Kochko and Hamon (1990) with some modifications as described earlier (Bhattacharya and Ranade, 2001). At least three to five independent DNA preparations were made from leaf tissues collected from each plant. The quantity and quality of DNA samples were estimated by comparing band intensities on agarose gel as well as by fluorometry

*Minisatellite, SSR, SLXY and RAPD Primers*: Four minisatellite core sequence primers and five SSR primers and three SLXY primers were custom synthesized from Bangalore Genei, Bangalore, India. The fifteen RAPD primers were procured from Qiagen Operon Technology Inc., Alameda, CA, USA. The sequences of all these primers as well as the

207F Bangla Nagaram Chinthalapudi (A.P.)a Jan. 2000 211M Kapoori Chillumurru -- Ditto -- Jan. 2000 213M Kapoori Tuni -- Ditto -- Jan. 2000 214M Kapoori Peddachapelli -- Ditto -- Jan. 2000 218M Kapoori Doddipatla -- Ditto -- Jan. 2000 219M Kapoori Chinnachapelli -- Ditto -- Jan. 2000 223F Bangla (U.P.) -- Ditto -- Jan. 2000 226M Kapoori Vuyyur (A.P.) -- Ditto -- Jan. 2000 234F Godi Bangla (Orrisa) -- Ditto -- Jan. 2000 235F Bangla (M.P.) -- Ditto -- Jan. 2000 239F Bangla Ramtek -- Ditto -- Jan. 2000 244F Kali Bangla (Assam) -- Ditto -- Jan. 2000 301M Kapoori Mhaisal Digraj (Maharashtra)b Jul. 2000 304M Kapoori Bolvad -- Ditto -- Jul. 2000 305M Kapoori Viddi -- Ditto -- Jul. 2000 306M Kapoori Karve -- Ditto -- Jul. 2000

**Source Time** 

(DyNA Quant 200, Pharmacia) using Hoechst 33258 as the fluorochrome.

annealing temperatures used in PCR with these primers are given in Table 2.

**Sample # Landrace Collection** 

Fig. 3. Photos of the female (top panel) and male (bottom panel) betelvines. The solid white arrows (top panel) and the solid black arrow (bottom panel) point to the female and male inflorescences respectively.
