**2. Materials and methods**

446 Ecosystems Biodiversity

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.

*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 stored at -70C till further use.

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

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


SPAR Profiles for the Assessment of Genetic Diversity Between

Primer Name

**SLXY primers** 

**ISSR method primers** 

**DAMD method primers** 

boldface are the optimum for that primer.

bases)

**RAPD Method primers** 

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 449

Annealing Temperature (C)

Primer Sequence (5'- 3') / (Length in numbers of

OPG-02 GGCACTGAGG / (10) **35**  OPG-03 GAGCCCTCCA / (10) **35**  OPG-08 TCACGTCCAC / (10) **35**  OPG-10 AGGGCCGTCT / (10) **35**  OPG-11 TGCCCGTCGT / (10) **35**  OPG-13 CTCTCCGCCA / (10) **35**  OPG-16 AGCGTCCTCC / (10) **35**  OPG-17 ACGACCGACA / (10) **35**  OPG-18 GGCTCATGTG / (10) **35**  OPG-19 GTCAGGGCAA / (10) **35**  OPH-04 GGAAGTCGCC / (10) **35**  OPH-06 ACGCATCGCA / (10) **35**  OPH-12 ACGCGCATGT / (10) **35**  OPH-18 GAATCGGCCA / (10) **35**  OPH-19 CTGACCAGCC / (10) **35** 

(GACA)4 GACAGACAGACAGACA / (16) 37, **40**, 45 (GATA)4 GATAGATAGATAGATA / (16) **30**, 33, 37 (GAA)6G GAAGAAGAAGAAGAAGAAG / (19) 41, 45, **48** (CA)8GC CACACACACACACACAGC / (18) **45**,48,53 (ACTG)4 ACTGACTGACTGACTG / (16) **38**, 41, 45

M-13 GAGGGTGGCGGTTCT / (15) 41, 45, **48** HVR(-) GCTCCTCCCTCCT / (13) **50**, 55 HBV GGTGTAGAGAGGGGT / (15) 41, 45, **48** 33.6 GGAGGTGGGCA / (11) **50**, 55

SLXY6 TGGACTTCCACTGGAATTCGAT / (21) 45, **50**, 55 SLXY7 ACTTGCAACGACTTCACTTTGAG / (25) 45, **50**, 55 SLXY8 ATCGAATTCCAGTGGAAGTCC / (22) 45, **50**, 55

Table 2. Various primers used for PCRs in present study. Annealing temperatures given in


a AICRP on Betelvine Center at Acharya NG Ranga Agricultural University, Bapatla, A. P. State. b AICRP on Betelvine Center at MPKV ARS at Kasbe Digraj, Sangli, Maharashtra State.

c AICRP on Betelvine Center at TNAU Sugarcane Research Station at Sirugamani, Tiruchirapalli, T. N. State.

d Betelvine Conservatory, at Betelvine Biotechnology Laboratory, NBRI, Lucknow, U. P. State.

e Mulberry accession number MI-0129 from an earlier NBRI collection of tissue for R&D work on mulberry.

Samples in shaded cells were not included in data analysis since their PCR profiles were not always consistent amongst replicate experiments.

Table 1. Betelvine landraces (gender set), *Piper hamiltonii* and mulberry included for the studies on PCR-based profiling are given below. In all cases, leaf tissue was harvested, washed and stored frozen as described. The source locations have been detailed as footnotes to the table. The sample number refers to the numbers assigned to the tissue samples while **M** or **F** suffixes to sample numbers indicate the appropriate gender as Male or Female respectively.

307M Kapoori Indapur -- Ditto -- Jul. 2000 310M Kapoori Arvi -- Ditto -- Jul. 2000 319F Calcutta Bangla -- Ditto -- Jul. 2000 323M Kapoori Maharashtra -- Ditto -- Jul. 2000 326M Kapoori Solapur -- Ditto -- Jul. 2000 409F Bangla Jal Sirigamani (T.N.)c Aug. 2000 410F Bangla Ramtek -- Ditto -- Aug. 2000 423F Bangla Jabalpur -- Ditto -- Aug. 2000 426F Bangla Desi -- Ditto -- Aug. 2000 427F Nav Bangla -- Ditto -- Aug. 2000 601F Kakair Sirugamani (T.N.)c Aug. 2003 602F Banchi Kodi -- Ditto -- Aug. 2003 603F Bangla Jal -- Ditto -- Aug. 2003 604F Kuljedu -- Ditto -- Aug. 2003 605M Kapoori Vasani -- Ditto -- Aug. 2003 606M Kapoori Bihar -- Ditto -- Aug. 2003 607F Deshawari -- Ditto -- Aug. 2003 608F SGM-1 -- Ditto -- Aug. 2003 609F Sreenivasa Nallur -- Ditto -- Aug. 2003 610F Bangla Ramtek -- Ditto -- Aug. 2003 612F Bangla -- Ditto -- Aug. 2003 613M Kapoori Chittikavata -- Ditto -- Aug. 2003 614M Tellaku Ponnur -- Ditto -- Aug. 2003 615F Shirpurkata -- Ditto -- Aug. 2003 616M Tellaku -- Ditto -- Aug. 2003 617M Tellaku Uttukuru -- Ditto -- Aug. 2003 618M Karpuri (T.N.) -- Ditto -- Aug. 2003 619M Sangli Kapoori -- Ditto -- Aug. 2003 620F Kalipatti -- Ditto -- Aug. 2003 621F Gach Pan -- Ditto -- Aug. 2003 PH Piper hamiltonii NBRI (Lucknow)d Aug. 2000 MO Mulberry NBRI (Lucknow)e Sep. 1999

a AICRP on Betelvine Center at Acharya NG Ranga Agricultural University, Bapatla, A. P. State. b AICRP on Betelvine Center at MPKV ARS at Kasbe Digraj, Sangli, Maharashtra State. c AICRP on Betelvine Center at TNAU Sugarcane Research Station at Sirugamani, Tiruchirapalli, T. N.

d Betelvine Conservatory, at Betelvine Biotechnology Laboratory, NBRI, Lucknow, U. P. State. e Mulberry accession number MI-0129 from an earlier NBRI collection of tissue for R&D work on

Samples in shaded cells were not included in data analysis since their PCR profiles were not always

Table 1. Betelvine landraces (gender set), *Piper hamiltonii* and mulberry included for the studies on PCR-based profiling are given below. In all cases, leaf tissue was harvested, washed and stored frozen as described. The source locations have been detailed as footnotes to the table. The sample number refers to the numbers assigned to the tissue samples while **M** or **F** suffixes to sample numbers indicate the appropriate gender as Male or Female

State.

mulberry.

respectively.

consistent amongst replicate experiments.


Table 2. Various primers used for PCRs in present study. Annealing temperatures given in boldface are the optimum for that primer.

SPAR Profiles for the Assessment of Genetic Diversity Between

repeated in replicated experiments were included in the analysis.

from the betelvine DNAs (data not shown).

calculated according to Powell *et al.* (1996).

individual assay and was calculated from:

for polymorphic markers (**DIp**) was calculated from:

included in the analysis.

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 451

5A) through 1.2% agarose gels in 0.5X TBE buffer, visualized and imaged using Nighthawk gel documentation system (pdi Inc., USA) after staining with ethidium bromide. Only distinct and well-separated bands repeated in replicated experiments were

*SPAR with SLXY primer (SLXY-PCR)*: The primers used for profiling, were synthesized from the prior known sequences specific to the X- and Y-chromosomes of the dioecious perennial plant *Silene latifolia* (Filatov *et al*., 2000). The reaction conditions were optimized for these primers also in the same way as for the RAPD primers. The final reactions included 100 ng of template, 20 pmoles of primer, 1.5 mM Mg2+ ions, 200 M each dNTP, 0.9 U *Taq* DNA polymerase in 1x assay buffer in a final volume of 15 l. All PCR were carried out in Air Thermal Cycler (Model ATC1605, Idaho Technology, Inc.). The Air Thermal Cycler was programmed to include pre-denaturation at 94°C for 60 s. This was followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 50°C for 30 s, and extension at 72°C for 45 s. The final cycle allowed an additional 240 s period of extension at 72°C. The PCR products were separated by electrophoresis (at constant current of 5A) through 1.4% agarose gels in 0.5X TBE buffer, visualized and imaged using Nighthawk gel documentation system (pdi Inc., USA) after staining with ethidium bromide. Only distinct and well-separated bands

*Data Analysis*: Data (fragment sizes of all amplification products, estimated from the gel by comparison with standard molecular weight marker, DNA double digested with *Hin*d III and *Eco*R I) were scored as discrete variables, using "1" to indicate presence and "0" to indicate absence of a band. A pair wise matrix of distances between genotypes was determined for the band data from each method using Jaccard coefficient (Jaccard, 1908) in the **FreeTree** program (ver. 0.9.1.5; Pavlicek *et al.* 1999). These pairwise distance data were used to compare the average distances estimated within and between the male and female betelvine landraces for each method. Additionally, a cumulative distance matrix, for the band data of all four methods considered together, was also computed separately to generate a single NJ tree after allowing a 1000 replicate bootstrap test using the same program. The tree was viewed, annotated and printed using **TreeView** (ver. 1.6.5; Page 2001). The robustness of the SPAR methods was tested in each case with a suitable non-*Piper* outgroup DNA included in the analysis, and in all cases outgroup was resolved as distinct

*Comparison of the different SPAR methods*: To determine the utility of each of the marker systems used, diversity index (**DI**), effective multiplex ratio (**E**) and marker index (**MI**) were

**DI** for genetic markers was calculated from the sum of the squares of the allele frequency:

**DIn = 1-∑pi**

where '**pi**' is the allele/band frequency of the 'ith' allele and '**n**' is the total number of loci. **DI**

**DIp = ∑ DIn / np**  where '**np**' is the number of polymorphic loci analyzed. Effective Multiplex Ratio (**E**) is the product of the fraction of polymorphic loci and the number of polymorphic loci for an

**2**

*SPAR with arbitrary sequence decamers (RAPD)*: Decamers from kits G and H (Operon Technologies Inc., Alameda, California, USA) were used as primers. DNA was amplified essentially following Williams *et al.* (1990). Initially a pilot experiment was carried out varying primer, template DNA and Mg++ ion concentrations. The final amplification reactions contained 1x buffer, 1.5 mM MgCl2, 200 M each dNTP, 10 pmoles primer, 0.6 U *Taq* DNA polymerase (Bangalore Genei, Bangalore, India) and 50 ng betelvine DNA template in a 10l reaction volume. The reaction was predenatured at 94°C for 1 min and thereafter cycled 44 times at 94°C for 15 s, 35°C for 20 s and 72°C for 45 s in the Air Thermal Cycler. Additionally a final extension cycle allowed incubation for 240 s at 72°C. The PCR products were separated by electrophoresis (at constant current of 5A) through 1% agarose gels in 0.5X TBE buffer, visualized and imaged using Nighthawk gel documentation system (pdi Inc., USA) after staining with ethidium bromide. Only distinct and wellseparated bands repeated in replicated experiments were included in the analysis.

*SPAR with SSR primers (ISSR-PCR)*: The PCR conditions were according to Gupta *et al.* (1994). A pilot experiment was carried out to determine the optimum annealing temperature for each primer in the range 3-10C lower than the denaturation temperature. 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 final reaction was carried out in 10 l volumes and contained 50 ng of template DNA, 10 pmoles of SSR primer, 200 M each dNTP, 1.5 mM Mg2+ ion concentration in suitable 1X assay buffer supplied along with the enzyme and 0.6 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 94°C for 60 s. This was followed by 30 cycles of denaturation at 94°C for 20 s, annealing at the optimized temperature for 30 s, and extension at 72°C for 45 s. The final cycle allowed an additional 240 s period of extension at 72°C. The reaction products obtained after PCR were analyzed on 1.2 or 1.8% agarose gels. The gel was stained in ethidium bromide and visualized and imaged on Nighthawk gel documentation system (pdi Inc., USA). Only distinct and well-separated bands repeated in replicated experiments were included in the analysis.

*SPAR with minisatellite primers (DAMD)*: The reaction was carried out essentially according to Zhou *et al.* (1997) for the primers 33.6, HBV and HVR(-) while for primer M13 the reactions were essentially according to Lorenz *et al.* (1995). The optimum annealing temperature was determined by carrying out DAMD at different annealing temperatures in the range 40C to 55C. The PCR parameters were as follows: First cycle of incubation at 94C for 60 s followed by 30 cycles of incubation at 94C for 20 s, at annealing temperature (41C in case of M13 primer and 50C in case of the other three primers) for 30 s and at 72C for 45 s. The final cycle allowed an additional incubation at 72C for 240 s. The reactions contained 100 ng template DNA, 40 pmoles of primer, 1.5 mM Mg2+ ions, 200 M each dNTP, 0.3 U *Taq* DNA polymerase in 1x assay buffer in a final volume of 15 l in case of the primers 33.6, HBV and HVR(-). In case of the M13 primer, the template, primer and enzyme and the reaction volumes were 60 ng, 10 pmoles, 0.6 Units and 10 l respectively. All amplification reactions were carried out in Air Thermal Cycler (Model ATC1605, Idaho Technology, Inc.) and the products were separated by electrophoresis (at constant current of

*SPAR with arbitrary sequence decamers (RAPD)*: Decamers from kits G and H (Operon Technologies Inc., Alameda, California, USA) were used as primers. DNA was amplified essentially following Williams *et al.* (1990). Initially a pilot experiment was carried out varying primer, template DNA and Mg++ ion concentrations. The final amplification reactions contained 1x buffer, 1.5 mM MgCl2, 200 M each dNTP, 10 pmoles primer, 0.6 U *Taq* DNA polymerase (Bangalore Genei, Bangalore, India) and 50 ng betelvine DNA template in a 10l reaction volume. The reaction was predenatured at 94°C for 1 min and thereafter cycled 44 times at 94°C for 15 s, 35°C for 20 s and 72°C for 45 s in the Air Thermal Cycler. Additionally a final extension cycle allowed incubation for 240 s at 72°C. The PCR products were separated by electrophoresis (at constant current of 5A) through 1% agarose gels in 0.5X TBE buffer, visualized and imaged using Nighthawk gel documentation system (pdi Inc., USA) after staining with ethidium bromide. Only distinct and well-

separated bands repeated in replicated experiments were included in the analysis.

in replicated experiments were included in the analysis.

*SPAR with SSR primers (ISSR-PCR)*: The PCR conditions were according to Gupta *et al.* (1994). A pilot experiment was carried out to determine the optimum annealing temperature for each primer in the range 3-10C lower than the denaturation temperature. 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 final reaction was carried out in 10 l volumes and contained 50 ng of template DNA, 10 pmoles of SSR primer, 200 M each dNTP, 1.5 mM Mg2+ ion concentration in suitable 1X assay buffer supplied along with the enzyme and 0.6 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 94°C for 60 s. This was followed by 30 cycles of denaturation at 94°C for 20 s, annealing at the optimized temperature for 30 s, and extension at 72°C for 45 s. The final cycle allowed an additional 240 s period of extension at 72°C. The reaction products obtained after PCR were analyzed on 1.2 or 1.8% agarose gels. The gel was stained in ethidium bromide and visualized and imaged on Nighthawk gel documentation system (pdi Inc., USA). Only distinct and well-separated bands repeated

*SPAR with minisatellite primers (DAMD)*: The reaction was carried out essentially according to Zhou *et al.* (1997) for the primers 33.6, HBV and HVR(-) while for primer M13 the reactions were essentially according to Lorenz *et al.* (1995). The optimum annealing temperature was determined by carrying out DAMD at different annealing temperatures in the range 40C to 55C. The PCR parameters were as follows: First cycle of incubation at 94C for 60 s followed by 30 cycles of incubation at 94C for 20 s, at annealing temperature (41C in case of M13 primer and 50C in case of the other three primers) for 30 s and at 72C for 45 s. The final cycle allowed an additional incubation at 72C for 240 s. The reactions contained 100 ng template DNA, 40 pmoles of primer, 1.5 mM Mg2+ ions, 200 M each dNTP, 0.3 U *Taq* DNA polymerase in 1x assay buffer in a final volume of 15 l in case of the primers 33.6, HBV and HVR(-). In case of the M13 primer, the template, primer and enzyme and the reaction volumes were 60 ng, 10 pmoles, 0.6 Units and 10 l respectively. All amplification reactions were carried out in Air Thermal Cycler (Model ATC1605, Idaho Technology, Inc.) and the products were separated by electrophoresis (at constant current of 5A) through 1.2% agarose gels in 0.5X TBE buffer, visualized and imaged using Nighthawk gel documentation system (pdi Inc., USA) after staining with ethidium bromide. Only distinct and well-separated bands repeated in replicated experiments were included in the analysis.

*SPAR with SLXY primer (SLXY-PCR)*: The primers used for profiling, were synthesized from the prior known sequences specific to the X- and Y-chromosomes of the dioecious perennial plant *Silene latifolia* (Filatov *et al*., 2000). The reaction conditions were optimized for these primers also in the same way as for the RAPD primers. The final reactions included 100 ng of template, 20 pmoles of primer, 1.5 mM Mg2+ ions, 200 M each dNTP, 0.9 U *Taq* DNA polymerase in 1x assay buffer in a final volume of 15 l. All PCR were carried out in Air Thermal Cycler (Model ATC1605, Idaho Technology, Inc.). The Air Thermal Cycler was programmed to include pre-denaturation at 94°C for 60 s. This was followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 50°C for 30 s, and extension at 72°C for 45 s. The final cycle allowed an additional 240 s period of extension at 72°C. The PCR products were separated by electrophoresis (at constant current of 5A) through 1.4% agarose gels in 0.5X TBE buffer, visualized and imaged using Nighthawk gel documentation system (pdi Inc., USA) after staining with ethidium bromide. Only distinct and well-separated bands repeated in replicated experiments were included in the analysis.

*Data Analysis*: Data (fragment sizes of all amplification products, estimated from the gel by comparison with standard molecular weight marker, DNA double digested with *Hin*d III and *Eco*R I) were scored as discrete variables, using "1" to indicate presence and "0" to indicate absence of a band. A pair wise matrix of distances between genotypes was determined for the band data from each method using Jaccard coefficient (Jaccard, 1908) in the **FreeTree** program (ver. 0.9.1.5; Pavlicek *et al.* 1999). These pairwise distance data were used to compare the average distances estimated within and between the male and female betelvine landraces for each method. Additionally, a cumulative distance matrix, for the band data of all four methods considered together, was also computed separately to generate a single NJ tree after allowing a 1000 replicate bootstrap test using the same program. The tree was viewed, annotated and printed using **TreeView** (ver. 1.6.5; Page 2001). The robustness of the SPAR methods was tested in each case with a suitable non-*Piper* outgroup DNA included in the analysis, and in all cases outgroup was resolved as distinct from the betelvine DNAs (data not shown).

*Comparison of the different SPAR methods*: To determine the utility of each of the marker systems used, diversity index (**DI**), effective multiplex ratio (**E**) and marker index (**MI**) were calculated according to Powell *et al.* (1996).

**DI** for genetic markers was calculated from the sum of the squares of the allele frequency:

$$\mathbf{DI\_n = 1} \mathbf{-} \sum \mathbf{p}^2$$

where '**pi**' is the allele/band frequency of the 'ith' allele and '**n**' is the total number of loci. **DI** for polymorphic markers (**DIp**) was calculated from:

#### **DIp = ∑ DIn / np**

where '**np**' is the number of polymorphic loci analyzed. Effective Multiplex Ratio (**E**) is the product of the fraction of polymorphic loci and the number of polymorphic loci for an individual assay and was calculated from:

SPAR Profiles for the Assessment of Genetic Diversity Between

bootstrapping was divided into two major clusters here also.

PCR were 0.25, 17.25 and 4.37 respectively (Table 3).

distinctly (NJ tree not shown).

landraces respectively.

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 453

bands were polymorphic across the betelvine landraces and only 5 bands were present in all landraces as well as *P. hamiltonii*. The Jaccard coefficients were computed and the highest (0.94) in female group was between 223 (Bangla U.P.) and 235 (Bangla M.P.) while the least (0.31) was between 409 (Bangla Jal) and 612 (Bangla). In male group the corresponding values were (0.97) between 219 (Kapoori Chinnachapelli) and 226 (Kapoori Vuyyur) and (0.47) between 307 (Kapoori Indapur) and 618 (Karpuri) and between 616 (Tellaku) and 304 (Kapoori Bolvad). Among female and male groups the highest was 0.50 between 618 (Karpuri), 410 (Bangla Ramtek), 223 (Bangla U.P.), 609 (Sreenivasa Nallur) and 619 (Sangli Kapoori), 608 (SGM-1) and 304 (Kapoori Bolvad) while the least (0.28) was between 616 (Tellaku) and 427 (Kapoori Bihar). The NJ tree after 1000 replicate bootstrapping revealed the betelvine landraces grouped together in two major clusters of the male and female landraces and here also as in the case of RAPD data 501 (*Piper Hamiltonii*) was separated out

**DAMD profiles of the gender set of betelvine landraces:** The DAMD profiles consisted of distinctly polymorphic banding patterns and the four DAMD primers resulted in a total of 68 polymorphic bands (19.00 bands per primer). Jaccard coefficients in the female group here revealed highest (0.83) was between 410 (Bangla Ramtek) and 610 (Bangla Ramtek). In male group the highest (0.95) was between 305 (Kapoori Viddi) and 326 (Kapoori Solapur). Between female and male, the highest value is 0.44 between 606 (Kapoori Bihar) and 604 (Kuljedu) and 617 (Tellaku Uttukuru). The distance data analyzed by NJ method using the FreeTree program, as described earlier revealed the NJ tree after 1000 replicate

**SLXY primer PCR profiles of the gender set of betelvine landraces:** PCR with SLXY primers used singly in the amplification reactions resulted in distinctly polymorphic banding patterns. A total of 60 bands (all polymorphic) were scored with the three SLXY primers with the average number of 17.25 bands per primer. The highest (0.91) between 207 (Bangla Nagaram) and 235 (Bangla M.P.) and least (0.22) between 234 (Godi Bangla), 426 (Bangla Desi) and 427 (Nav Bangla) coefficients were computed in the female group. In male group the corresponding values are 1.0 between 218 (Kapoori Doddipatla) and 213 (Kapoori Tuni); 0.26 between 619 (Sangli Kapoori) and 614 (Tellaku Ponnur). Between female and male, the highest value is 0.45 between 617 (Tellaku Uttukuru) and 606 (Kapoori Bihar) and least 0.95 was between 427 (Nav Bangla) and 619 (Sangli Kapoori). The NJ tree after 1000 replicate bootstrapping reveals *Piper Hamiltonii* (501) were clearly separated from the rest of the betelvine landraces that were in turn divided into two major clusters of male and female

**Comparison of the different PCR methods used:** Four different PCR-based methods were used to assess diversity and to distinguish between the genders. In order to determine the utility of each of these PCR-based methods used, a comparative statistical assessment was done according to Powell *et al*., (1996). Diversity Index (**DI**) or Heterozygosity Index, Marker Index (**MI**) and Effective Multiplex Ratio (**E**) were calculated in case of each of the methods used as described in the materials and methods. By RAPD method **DI** value was 0.29, **E** value was 17.13 and **MI** value was 4.91. For the same set of landraces with ISSR-PCR, **DI** was 0.33, **E** was 16.2 and **MI** was 5.3. When DAMD primers were used for the analysis, **DI** value was 0.23, **E** was 19.0 and **MI** was 4.4. The corresponding values for the SLXY primer

#### **E = np(np / n)**

**MI** is defined as the product of the average diversity index for polymorphic bands in any assay and the effective multiplex ratio (**E**) for that assay. It was calculated as:

#### **MI = DIp x E**

The Mantel matrix correspondence test (Mantel, 1967) was used to compare distance matrices for each SPAR method with the help of the program **Mantel 2** (Liedloff, 1999).

#### **3. Results**

**Optimization of the different PCRs:** Screening of the genotypes and various optimization experiments was carried out so as to identify the reaction parameters and conditions including concentrations of template, Mg2+ ions, primers and dNTPs used as well as the annealing temperatures in the PCRs that enabled the most repeatable results. Likewise, consistency of the profiles was judged by using duplicate or triplicate template samples for the same primers such that all the prominent bands were consistently produced with each of the template replicates. In all 22 male and 24 female betelvine variety DNAs were used in the present study along with *Piper hamiltonii* and mulberry DNAs as outgroups. The last named mulberry DNA outgroup was used just to determine the robustness of the PCR reactions and data from this was not scored for the analysis. On the basis of the preliminary optimizations (data not shown) template DNAs of male varieties, 211, 301 and 310 (Table 1) were excluded from data analysis since profiles with these were not consistent with all primers and all replicates tested.

**RAPD analysis of the gender set of betelvine landraces:** A total of 15 primers (Table 2) were used for RAPD analysis and all the primers resulted in discrete amplification products. Three landraces 211, 301 and 310 failed to give discrete profiles with some of the primers so data for these landraces was not scored from the profiles for final calculations and analysis**.** The RAPD data for all 15 primers were considered cumulatively and included a total of 219 bands (17.13 bands per primer) for the analysis of the relationship amongst the betelvine landraces. Of these some 98% bands were polymorphic and only four bands were present in all betelvine and *Piper hamiltonii* DNAs. Jaccard coefficients were computed from the band data and the highest (0.86) in female group was between two Bangla Ramtek accessions 239 (from Chinthalapudi) and 610 (from Sirugamani) while the least (0.22) was between 604 (Kuljedu) and 620 (Kallipati) and 427 (Nav bangla). The corresponding values in case of the male landraces were 0.96 between 213 (Kapoori Tuni) and 214 (Kapoori Peddachapelli) and 0.23 between 307 (Kapoori Indapur) and 218 (Kapoori Doddipatla). Between male and female, the highest (0.40) was between 326 (Kapoori Solapur) and 612 (Bangla) whereas least (0.18) was between both 305 (Kapoori Viddi) and 326 (Kapoori Solapur) and 427 (Nav Bangla). The distance data computed from the Jaccard coefficients were analyzed further by NJ method, using the FreeTree program, as described earlier in the materials and methods, to describe the relationship between betelvine landraces. The NJ tree after 1000 replicate bootstrapping revealed two major clusters respectively for the female and male landraces and the out group 501 (*Piper Hamiltonii*) was separated from these two clusters (NJ tree not shown).

**ISSR-PCR profiles of gender set of betelvine landraces:** Here five primers resulted in discrete patterns of 73 closely spaced bands (16.20 bands per primer). Of these some 93%

**E = np(np / n) MI** is defined as the product of the average diversity index for polymorphic bands in any

**MI = DIp x E**  The Mantel matrix correspondence test (Mantel, 1967) was used to compare distance matrices for each SPAR method with the help of the program **Mantel 2** (Liedloff, 1999).

**Optimization of the different PCRs:** Screening of the genotypes and various optimization experiments was carried out so as to identify the reaction parameters and conditions including concentrations of template, Mg2+ ions, primers and dNTPs used as well as the annealing temperatures in the PCRs that enabled the most repeatable results. Likewise, consistency of the profiles was judged by using duplicate or triplicate template samples for the same primers such that all the prominent bands were consistently produced with each of the template replicates. In all 22 male and 24 female betelvine variety DNAs were used in the present study along with *Piper hamiltonii* and mulberry DNAs as outgroups. The last named mulberry DNA outgroup was used just to determine the robustness of the PCR reactions and data from this was not scored for the analysis. On the basis of the preliminary optimizations (data not shown) template DNAs of male varieties, 211, 301 and 310 (Table 1) were excluded from data analysis since profiles with these were not consistent with all

**RAPD analysis of the gender set of betelvine landraces:** A total of 15 primers (Table 2) were used for RAPD analysis and all the primers resulted in discrete amplification products. Three landraces 211, 301 and 310 failed to give discrete profiles with some of the primers so data for these landraces was not scored from the profiles for final calculations and analysis**.** The RAPD data for all 15 primers were considered cumulatively and included a total of 219 bands (17.13 bands per primer) for the analysis of the relationship amongst the betelvine landraces. Of these some 98% bands were polymorphic and only four bands were present in all betelvine and *Piper hamiltonii* DNAs. Jaccard coefficients were computed from the band data and the highest (0.86) in female group was between two Bangla Ramtek accessions 239 (from Chinthalapudi) and 610 (from Sirugamani) while the least (0.22) was between 604 (Kuljedu) and 620 (Kallipati) and 427 (Nav bangla). The corresponding values in case of the male landraces were 0.96 between 213 (Kapoori Tuni) and 214 (Kapoori Peddachapelli) and 0.23 between 307 (Kapoori Indapur) and 218 (Kapoori Doddipatla). Between male and female, the highest (0.40) was between 326 (Kapoori Solapur) and 612 (Bangla) whereas least (0.18) was between both 305 (Kapoori Viddi) and 326 (Kapoori Solapur) and 427 (Nav Bangla). The distance data computed from the Jaccard coefficients were analyzed further by NJ method, using the FreeTree program, as described earlier in the materials and methods, to describe the relationship between betelvine landraces. The NJ tree after 1000 replicate bootstrapping revealed two major clusters respectively for the female and male landraces and the out group 501 (*Piper Hamiltonii*)

**ISSR-PCR profiles of gender set of betelvine landraces:** Here five primers resulted in discrete patterns of 73 closely spaced bands (16.20 bands per primer). Of these some 93%

assay and the effective multiplex ratio (**E**) for that assay. It was calculated as:

**3. Results** 

primers and all replicates tested.

was separated from these two clusters (NJ tree not shown).

bands were polymorphic across the betelvine landraces and only 5 bands were present in all landraces as well as *P. hamiltonii*. The Jaccard coefficients were computed and the highest (0.94) in female group was between 223 (Bangla U.P.) and 235 (Bangla M.P.) while the least (0.31) was between 409 (Bangla Jal) and 612 (Bangla). In male group the corresponding values were (0.97) between 219 (Kapoori Chinnachapelli) and 226 (Kapoori Vuyyur) and (0.47) between 307 (Kapoori Indapur) and 618 (Karpuri) and between 616 (Tellaku) and 304 (Kapoori Bolvad). Among female and male groups the highest was 0.50 between 618 (Karpuri), 410 (Bangla Ramtek), 223 (Bangla U.P.), 609 (Sreenivasa Nallur) and 619 (Sangli Kapoori), 608 (SGM-1) and 304 (Kapoori Bolvad) while the least (0.28) was between 616 (Tellaku) and 427 (Kapoori Bihar). The NJ tree after 1000 replicate bootstrapping revealed the betelvine landraces grouped together in two major clusters of the male and female landraces and here also as in the case of RAPD data 501 (*Piper Hamiltonii*) was separated out distinctly (NJ tree not shown).

**DAMD profiles of the gender set of betelvine landraces:** The DAMD profiles consisted of distinctly polymorphic banding patterns and the four DAMD primers resulted in a total of 68 polymorphic bands (19.00 bands per primer). Jaccard coefficients in the female group here revealed highest (0.83) was between 410 (Bangla Ramtek) and 610 (Bangla Ramtek). In male group the highest (0.95) was between 305 (Kapoori Viddi) and 326 (Kapoori Solapur). Between female and male, the highest value is 0.44 between 606 (Kapoori Bihar) and 604 (Kuljedu) and 617 (Tellaku Uttukuru). The distance data analyzed by NJ method using the FreeTree program, as described earlier revealed the NJ tree after 1000 replicate bootstrapping was divided into two major clusters here also.

**SLXY primer PCR profiles of the gender set of betelvine landraces:** PCR with SLXY primers used singly in the amplification reactions resulted in distinctly polymorphic banding patterns. A total of 60 bands (all polymorphic) were scored with the three SLXY primers with the average number of 17.25 bands per primer. The highest (0.91) between 207 (Bangla Nagaram) and 235 (Bangla M.P.) and least (0.22) between 234 (Godi Bangla), 426 (Bangla Desi) and 427 (Nav Bangla) coefficients were computed in the female group. In male group the corresponding values are 1.0 between 218 (Kapoori Doddipatla) and 213 (Kapoori Tuni); 0.26 between 619 (Sangli Kapoori) and 614 (Tellaku Ponnur). Between female and male, the highest value is 0.45 between 617 (Tellaku Uttukuru) and 606 (Kapoori Bihar) and least 0.95 was between 427 (Nav Bangla) and 619 (Sangli Kapoori). The NJ tree after 1000 replicate bootstrapping reveals *Piper Hamiltonii* (501) were clearly separated from the rest of the betelvine landraces that were in turn divided into two major clusters of male and female landraces respectively.

**Comparison of the different PCR methods used:** Four different PCR-based methods were used to assess diversity and to distinguish between the genders. In order to determine the utility of each of these PCR-based methods used, a comparative statistical assessment was done according to Powell *et al*., (1996). Diversity Index (**DI**) or Heterozygosity Index, Marker Index (**MI**) and Effective Multiplex Ratio (**E**) were calculated in case of each of the methods used as described in the materials and methods. By RAPD method **DI** value was 0.29, **E** value was 17.13 and **MI** value was 4.91. For the same set of landraces with ISSR-PCR, **DI** was 0.33, **E** was 16.2 and **MI** was 5.3. When DAMD primers were used for the analysis, **DI** value was 0.23, **E** was 19.0 and **MI** was 4.4. The corresponding values for the SLXY primer PCR were 0.25, 17.25 and 4.37 respectively (Table 3).

SPAR Profiles for the Assessment of Genetic Diversity Between

respectively).

**4. Discussion** 

general and functional dioecy in particular.

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 455

Another parameter for comparing the different profiling methods is to correlate the respective distance matrices (computed from the Jaccard coefficients) by a Mantel test. Such a correlation analysis was carried out (Table 4) and resulted in highly significant correlation (standard variate **g > p 0.005** value) in all cases. However, for the four methods the highest correlation was between RAPD and ISSR distance matrices (**r** = 0.8432, Table 4). This clearly indicates that relative distances of the landraces to each other were almost similarly estimated by both RAPD and ISSR methods. On the other hand, in case of the DAMD method, it was less strongly correlated (**r** = 0.5009, 0.4619 and 0.3850) to the other three methods (RAPD, ISSR and SLXY respectively). The correlation between RAPD and ISSR with SLXY primer data was almost equally strong in both cases (**r** = 0.7853, 0.7854

Dioecy is a widespread condition in flowering plants. Despite their recent evolutionary origin, 6% of the 240,000-angiosperm species are dioecious and 7% of 13,000 genera of angiosperms include dioecious species, suggesting that it has arisen many times during flowering plant evolution (Renner and Ricklefs, 1995). Dioecy is correlated with perennial climbing growth, wind or water pollination and has a preponderence in tropical flora. Natural selection, development of complex physiological and morphological traits, male fitness, ecological context, sex ratio, phylogenetic perspective and nuclear-cytoplasmic gynodioecy are some of the factors affecting distribution of dioecy. The plant taxa can offer insights into level of dimorphism that exists prior to the evolution of complete dioecy and the tradeoffs or constraints faced by hermaphrodites. The betelvine is one such interesting dioecious plant with an almost obligate vegetative propagation, lianaceous habit and perennial growth that provides a good system for studying molecular aspects of dioecy in

The study with PCR profiles was the first step towards resolving gender differences, if any, in betelvines. In an earlier study with 53 landraces that included only a few landraces with known genders, the RAPD method had provided a distinction between male and female betelvines (Verma *et al.*, 2004). In the present study with a larger sampling specifically amongst the betelvines with known genders, the bootstrap NJ tree for the RAPD data clearly differentiated the male and female landraces into two separate broad clusters, thereby supporting our original grouping of the landraces in terms of male and female vines based on known or actual flowering data. Banerjee *et al.* (1999) have reported a similar study of RAPD profile variation in another dioecious species, *Piper longum* L. and have further shown that at least two RAPD fragments were consistently associated with male plants. The clear separation of the betelvines on the basis of gender provides important leads for the identification and development of gender specific primers and probes. This work, however, has an important caveat that the economically most important product of the betelvines are the leaves and leaves of both male and female vines have a market value. Therefore the gender specific detection and or diagnosis will have only an academic value in case of betelvines unlike that in plants such as Papaya (Deputy *et al.*, 2002; Ma *et al.*, 2004). ISSR-PCR has been successfully utilized to distinguish gender or gender-specific markers in dioecious plants like hops and datepalm (Jakse et al. 2008; Younis et al. 2008). In the present


Table 3. The comparison of different PCR methods for the assessment of genetic diversity in the gender set of betelvine landraces.


**\*\*** Highly significant values (Critical value **p**0.005 = 2.575)

**+++**: High correlation

**<sup>+</sup>**: Moderate or low correlation

Table 4. Correlations among the distance matrices for the RAPD, ISSR, DAMD and SLXY primer PCR band data in case of the gender based set of the betelvine landraces. A significant, (p = 0.005), standard normal variate **(g)** was obtained among the matrices generated by all the four methods in all possible pairs of matrix comparison.

Another parameter for comparing the different profiling methods is to correlate the respective distance matrices (computed from the Jaccard coefficients) by a Mantel test. Such a correlation analysis was carried out (Table 4) and resulted in highly significant correlation (standard variate **g > p 0.005** value) in all cases. However, for the four methods the highest correlation was between RAPD and ISSR distance matrices (**r** = 0.8432, Table 4). This clearly indicates that relative distances of the landraces to each other were almost similarly estimated by both RAPD and ISSR methods. On the other hand, in case of the DAMD method, it was less strongly correlated (**r** = 0.5009, 0.4619 and 0.3850) to the other three methods (RAPD, ISSR and SLXY respectively). The correlation between RAPD and ISSR with SLXY primer data was almost equally strong in both cases (**r** = 0.7853, 0.7854 respectively).
