(PROFORMA FOR SUBMISSION OF FINAL REPORT OF RESEARCH PROJECTS)

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1 1 (PROFORMA FOR SUBMISSION OF FINAL REPORT OF RESEARCH PROJECTS) Part I: General Information 800 Project Code 8001 Institute project code No. Gen VIII (813) 8002 ICAR project code No. 801 Name of the Institute and Division 8011 Name and Address of Institute: Indian Institute of Spices Research, Calicut 8012 Name of Division/Section: Crop Improvement 8013 Location of the Project: IISR, Calicut 802 Project Title: Molecular characterization and in vitro propagation of Myristica spp. 803 Priority Area 8031 Research Approach: Applied Research (01) 804 Specific Area: Biotechnology 805 Duration of Project: Four years 8051 Date of start: Aug Date of completion: Nov Total Cost/expenditure incurred: 6.85 lakhs (Give reasons for variation from original estimated cost) 807. Executive summary The germplasm repository at IISR, harbours different collections of cultivated, wild, endangered and related species of Myristica. The project aims at molecular level characterization of this valuable germplasm through RAPD. Besides, attempts shall be made for standardizing tissue culture protocols in Myristica fragrans. Attempts are also made for identifying sex specific markers from Myristica fragrans. 808 Key words Myristica, RAPD, micropropagation Part II: Investigator Profile (Please identify clearly changes, if any in project personnel) 810 Principal Investigator: 8101 Name: Dr. Sheeja T.E Designation: Scientist Senior Scale (Biotechnology) 8103 Division/Section: Division of Crop Improvement

2 Location: IISR 8105 Institute Address: Indian Institute of Spices Research, Calicut Co-Investigator: 8111 Name: Dr. B. Krishnamoorthy 8112 Designation: Head, Division of Crop Improvement 8113 Division/Section: Division of Crop Improvement 8114 Location: Calicut 8115 Institute Address: Indian Institute of Spices Research, Calicut Part III: Technical Details 820 Introduction and Objectives 8201 Project Objectives Optimization of micropropagation protocols in Myristica fragrans. Molecular characterization and identification of phylogenetic relatedness among the cultivated and wild species through RAPD using random primers of OPERON Identification of sex specific markers 8202 Background information and importance of the project Myristica fragrans is a dioecious crop. It is difficult to distinguish the sex of nutmeg trees until flowering has taken place i.e., within 5-8 years. This leads to great economic loss since male and bisexual trees are undesirable due to poor yield. The problem has been circumvented by vegetative propagation of female trees. A very high yielding compact type of nutmeg viz. IISR, Viswashree had been released. Besides, a large number of elite trees have been identified both in the germplasm and farmer s field. Hence for large scale multiplication of these promising lines/released varieties, epicotyl grafting alone may not be sufficient as to meet the heavy demand for planting materials due to paucity of scions. Hence protocols for mass production have to be standardized for generating large number of female plantlets in vitro.

3 3 Germplasm repository at IISR, Calicut possesses a large collection of Myristica fragrans and other Myristica sp. obtained from diverse locations. Even though all the different species are not commercially exploited, still it is felt that the germplasm is of utmost importance with regard to conservation of biodiversity. The germplasm has been characterized through conventional approaches, however till date no information exists for the molecular level identification of the different Myristica species. The phylogenetic relatedness of the different species is also not analysed, which will help in adopting appropriate breeding strategies for improvement of this valuable spice Project Technical Profile Technical Programme: (Indicate briefly plan of procedure, techniques, instruments and special materials, organisms and special environment etc.) Methodology Plant material used for optimization of DNA isolation and PCR protocol was that of a released variety of Myristica fragrans viz., IISR Viswashree and nine wild species of Myristica viz., M. malabarica, M. fatua, M. beddomeii, M. prainii, M. andamanica, Knema andamanica, Gymnocranthera canerica, M. amygdalina and an unidentified species of Myristica maintained in the germplasm repository of IISR at Peruvannamuzhi, Calicut. Attempts to optimize in vitro protocols involved different media like MS, SH, WPM with varying levels of different phytohormones and other physical conditions. Attempts made to optimize in vitro propagation protocols in M. fragrans for optimizing various components like media, phytohormones, adjuvents, gelling agents etc. for obtaining callus induction and regeneration response DNA was isolated as per a modified protocol in the lab. Fresh leaves were collected and stored in iceboxes until reaching the lab after which, they were dipped in liquid nitrogen and stored at 80 0 C until extraction of DNA. For optimization experiments the composition of DNA extraction buffer used was as described by Doyle and Doyle (1990) with CTAB at 1, 2, 3, 4% and SDS at 0.5, 1, 1.5, 2% concentrations. The ph of the buffer was adjusted to either 8 or 9. The DNA isolation was conducted

4 4 following the CTAB protocol (Murray and Thompson 1980) with modifications, described schematically below: DNA Purification DNA purification was done by adding RNase (100 µg/ml) to the DNA solution and incubating for one hour at 37 o C. Equal volume of buffer saturated phenol:chloroform: isoamyl alcohol (25:24:1) was added, mixed properly and centrifuged at 12,000 g for 10 min at 4 o C. Supernatant was extracted with an equal amount of chloroform:isoamyl alcohol (24:1). Sodium acetate (3 M; 1/10 V) and ice-cold isopropanol was added and kept at 20 o C for 30 min. Centrifugation was performed at g for 10 min at room temperature. Precipitated DNA was washed with 70% ethanol, air dried, suspended in 100 µl of water and reprecipitated using 100% ethanol, centrifuged at 12,000 g for 10 minutes and washed in 70% ethanol, air dried and stored. The purity of the DNA sample was determined by measuring absorbance at A 260 nm in a UV spectrophotometer and A 260/ A 280 ratio was evaluated. Molecular weight and concentration of the DNA was estimated using agarose gel electrophoresis on 0.8% agarose and visualized by ethidium bromide staining against Human Genomic DNA. The quality of DNA was ascertained through restriction digestion using ECOR 1 and Hind III (Banglore Genei, India) both by single and double digestion as per manufacturer s protocol. Optimisation of PCR Parameters RAPD was performed as per Williams et al., (1990). The reaction was carried out in a thermal cycler (MJ Research). The amplification profiles involved an initial denaturation at 93 C for 3 min, 40 cycles of denaturation at 93 C for 1 min, annealing at 40 C for 1 min, extension at 72 C for 1 minute and a final extension at 72 C for 10 min. Amplification products were resolved on a 2% agarose gel stained with ethidium bromide and electrophoresed at 80 V for 2.5 hours. The gels were photographed and visualized in a Gel documentation system (Alpha Imager, USA) and raw gel images were recorded through molecular Analyst Software/PC version 99.04).

5 5 Factors like DNA concentration (10, 20, 30, 40 and 50 ng of DNA in 25 µl reaction volume), Mg +2 (1-10 mm), Taq DNA Polymerase (0.5, 1.0, 1.5 U in combination with 0.1, 0.2, 0.3, 0.5, 1.0, 1.25 and 1.5 mm dntp) and three brands of Taq DNA polymerase (Bangalore Genei; and Biogene, USA viz., Sure Taq and Doctor Taq), annealing temperature (37, 40, 45, 50 0 C), different thermocyclers (MJ Research, PTC 200 and Gene Amp PCR systems 9700) and primers singly and in combination were tried for optimization in Myristica sp. All the PCR optimization experiments were repeated twice using primers OPA 01 and OPC 06 (OPERON technologies, USA). In case of the experiments involving primer pairs, OPA 03 and OPA 05 were used as the second pair. About 45 random primers were screened and 13 that gave good amplification were selected for analysis of genetic relationship among the accessions. ISSR PCR was done according to Johnson et al. (2006). Phylogenetic analysis The banding patterns were recorded using a gel documentation system (Biorad) and image profiles and molecular weight of each band were determined by Alpha Imager. Electrophoretic patterns were reconstructed according to the presence or absence of clear, visible and reproducible bands (Williams et al., 1990). The most intense monomorphic band from each accession with each primer was used as reference to calibrate the different lanes for amount of DNA present. When there was no monomorphic band, the band with highest frequency in each sample was used for calibration. Within each lane, bands were scored present if their intensity was at least 10% that of monomorphic reference band within that lane. Markers were scored as present (1) and absent (0) for each accession. Band sharing data were used to calculate genetic similarities based on the Jaccard Coefficient (Jaccard, 1908) and UPGMA algorithm was employed to determine the genetic relationships. All analyses were performed using NTSYS-pc software (version 1.7) Total man months involvement of component project workers Principal Scientist: 2 X4= 8 Scientist: 10.25X4= 41

6 6 822 Final Report on the Project Detailed report of the project containing all relevant data with a brief summary of results. The total duration of the project was four years commencing from Aug 2004 to Nov The objectives of the project could be completed within the time frame specified. The results of the study warrants further work on sex specific markers identified. Confirmation of sex specific nature of these bands and identification of few more sex specific markers is needed. The detailed report follows: 1. Optimization of in vitro culture protocols in M. fragrans Attempts were made to optimize in vitro propagation protocols in M. fragrans. Callus induction could be achieved from mace explants on MS medium supplemented with 2mg/l BAP or Kinetin in presence of 0.5 mg/l NAA and % charcoal. From nodal explants bud break could be obtained in 3% cultures on both SH and WPM media. The growth and survival of buds were found to be better in presence of 2 mg/l BAP and 0.5 mg/l NAA in SH media. However, the buds failed to survive on any of the different media combinations tried. Callus induced from immature mace showed very poor regeneration ability and became compact and dry after prolonged maintenance on media. High rate of contamination due to fungus is found to affect the survival of in vitro cultures. The results of the study indicate that sterile cultures can be established from nodal explants. Callus induction and bud break is achievable in nutmeg. A B Fig 1. A: Callus induction of young immature mace explants on MS supplemented with 2 mg/l BAP and 0.5 mg/l NAA. B: Callus induction observed all over the explant

7 7 A B Fig 2. A&B: Initiation of bud break from nodal explants in Myristica fragrans Conclusions Callus induction could be achieved from young mace explants of immature fruits of M. fragrans. Bud break from nodal explants achieved in M. fragrans Axenic cultures could be established in M. fragrans This is a highly recalcitrant plant species, difficult to regenerate from callus High rate of contamination is observed that leads to difficulty in establishment of culture in this species due to systemic fungus Phenolic exudation is observed that hampers establishment of cultures in vitro 2. Optimization of DNA Isolation and PCR Parameters in Myristica sp. and Related Genera for RAPD and ISSR Analysis An efficient protocol for isolation of DNA from wild and related genera of Myristica rich in polysaccharides and polyphenols was developed (Fig 3-1a &1b). The protocol utilizes CTAB (3%), 1.5% PVP and 0.3% β-mercaptoethanol for isolation and RNase and phenol chloroform extraction for purification. DNA yield from very young leaves of Myristica was poor with large amounts of RNA. DNA isolated from older leaves showed low

8 8 recovery and contamination with protein. Third leaves from the shoot tip were found to give good quality of DNA in comparison to young shoot tips and older leaves with A 260 /A 280 ratio as The DNA obtained was of high molecular weight (~23 Kb), showed no shearing and gave clear bands. DNA yield from different treatments ranged from 2.4 to 150 ng/g of leaf tissue for CTAB and 4-30 ng/g in case of SDS as per the original protocols that showed very low yields of DNA with degradation and more than one bands on the gel (Figure 4 1a). Among the different concentrations of CTAB used (1%, 2%, 3%, 4%) at ph 8 and ph 9, it was found that CTAB of all concentration at ph 9 gave good DNA yield without RNA contamination. The DNA yield obtained from CTAB at 2%, 3% and 4% concentrations was 40, 150 and 125 ng respectively with A 260 /A 280 ranging from DNA yield in presence of SDS was lower than that of CTAB at both the ph tried. The yield and quality of DNA obtained from wild and related genera of Myristica is listed in the Table 1; Figure 3-1c. The purity of DNA was further confirmed upon restriction digestion using enzymes ECOR I and Hind III singly and in combination and a characteristic smear was obtained on agarose (Fig 4-2). Regarding optimization of PCR parameters, DNA concentration of 20 ng in 25µl reaction volume was found to give distinct scorable bands. The DNA was tested for amplification using random primers and ISSR primers. In case of wild species, purification using RNase and phenol- chloroform extraction (twice) was inevitable for successful amplification by PCR, while in accessions of Myristica fragrans it was not essential. The DNA isolated as per the above protocol was successfully amplified using 15 RAPD and seven ISSR primers as represented in Figure 4 (4a, 4b). The stability of DNA on long term storage and complete digestion with restriction enzymes and amplification by RAPD and ISSR showed absence of polysaccharides. Successful double and single digestion with ECOR 1 and Hind III indicates that the present method is suitable for DNA isolation involving sensitive operations like AFLP, RFLP, Southern blotting etc. DNA concentration of 20 ng/reaction gave distinct scorable bands in Myristica. In coconut better amplification is reported at 20 ng. Annealing temperature of 37 0 C is commonly used for RAPD. But in the case of Myristica it was observed that 45 0 C gave

9 9 best results. Optimum annealing temperatures was high in Myristica, however, very high temperature above 45 0 C lead to diffusion and disappearance of bands. MgCl 2 concentration of 1.5 mm is commonly used. In the present study no amplification was observed at this concentration, whereas 2-4 mm gave good amplification. In coconut also, MgCl 2 of 3 and 4 mm gave optimum intensity of bands. Number of bands was found to decrease along with a reduction in clarity with increasing levels of MgCl 2. dntp concentration is a crucial factor determining clarity and reproducibility of banding patterns. dntp levels were optimized in conjunction with that of Taq DNA polymerase to avoid artifacts due to their interaction. In the standard PCR protocol, each dntp concentration is 2 mm. However lower concentrations are known to give higher fidelity and specificity. Although amplification was observed with all these concentrations of enzyme and dntp, 0.5 U enzyme and 3 mm dntp gave most intense bands with good resolution (Fig 4-3). While using primer pairs, the occurrence of additional bands and disappearance of bands may be attributed to the competitive nature of primer target site selection. Fragments with annealing sites for 2 different primers at the ends do not form hairpin structures and these primers will not be out competed by internal hairpin formation to be occurring in fragments having identical primers at both ends. It is advisable to use more single primers than using primer pairs. Taq polymerases of different brands were used for amplification. The observation denotes that amplification profiles vary with the Taq DNA polymerase used. Earlier workers, have also reported that different polymerase gave different profiles. It is observed that for obtaining consistency in banding profiles and for better comparison of experiments, the same Taq DNA polymerase should be used. Sure Taq (Biogene, USA) though cheap, gave good results in case of Myristica. RAPD and ISSR analysis has revolutionized the genetic analysis of cultivars, varieties and breeding lines mainly due to reasons like ease and less time consuming analysis, non requirement of prior knowledge of sequence data and availability of a large number of primers. They are considered to be good tools for assessing genetic diversity at

10 10 molecular level and has been employed in phylogenetic studies in various plant species especially in closely related individuals as distinguishing tags for genotypes and varieties. The procedure described here is simple, efficient and economical and works well for extracting high quality DNA from elite accessions and wild species of Myristica and should be widely applicable for DNA analysis of large populations. This protocol may also be extended to other related species of Myristica. This is the first report of isolation of genomic DNA and amplification by PCR from wild and related genera of Myristia. PCR protocols were optimised involving experimental parameters such as DNA concentration (20 ng), MgCl 2 (2 mm), dntp (300 µm), different brands as well as concentration of Taq polymerase, annealing temperature (45 0 C), combination of random primers and single primer, amplification of DNA without RNase and phenol chloroform treatment (Fig 3 A-G). Forty five primers were screened and 15 gave good amplification. dntps at 0.3 mm in presence of Taq at 0.5 U and 2 mm MgCl 2 was best. 45 o C annealing temperature is optimum in nutmeg. Sure Taq of Biogene was found to be best. Single primers yielded better polymorphism than combination of primers. A method was also developed for PCR amplification in nutmeg without RNase and phenol chloroform treatment, which is very cheap and less time consuming and applicable to both RAPD and ISSR (Figure 4 (4a, 4b). Table 1. Yield and purity of DNA from Myristica species and related genera No Species Yield of DNA (µg/g fresh leaf OD value A 260 /A M. fragrans M. beddomeii M. malabarica M. prainii M. fatua M. andamanica K. andamanica M. amygdalina Unidentified sp Gymnocranthera canerica

11 11 A B C D

12 12 E F G Fig 3 A-G. Optimisation of PCR conditions for Taq polymerase, dntp and Thermocyclers

13 Fig 4 (1a, 1b). Agarose gel of total genomic DNA isolated from M. fragrans using different concentrations of CTAB (a) original protocol, (b) present protocol. Lanes 1-4: DNA obtained using 1, 2, 3, 4 % CTAB extraction buffer. Fig 4 1c: Agarose gel of total genomic DNA isolated from different wild and related genera of Myristica. 1- M. fragrans, 2- M. malabarica, 3- M. beddomeii, 4- M. prainii 5- M. fatua, 6- M. andamanica, 7- K. andamanica, 8- M. amygdalina, 9- Unidentified species, 10- Gymnocranthera canerica Fig 4 (2): Agarose gel electrophoresis of digested genomic DNA isolated as per the new protocol from M. fragrans (1-3) and M. fatua (4-6). M- 1 Kb ladder; Lanes 1, 4: ECORI, Lanes 2, 5: Hind III, Lanes 3, 6: double digestion. Fig 19 (3): PCR amplification of 20 ng DNA at varying concentrations of dntp and Taq DNA polymerase in M. fragrans. Lanes 1-3: dntp 0.15 mm with 0.5, U of Taq, Lanes 4-6: dntp 0.2 mm with 0.5, U of Taq, Lanes 7-9: dntp 0.3 mm with 0.5, U of Taq, Lanes 10-12: dntp 0.4 mm with 0.5, U of Taq. Fig 4 (4a, b): Representative amplification pattern of DNA from ten species of Myristica. a: RAPD profile obtained with OPB 20 primer. b: ISSR profile obtained using ISSR 11 primer. M- 1 Kb ladder, 1- M. fragrans, 2- M. malabarica, 3- M. beddomeii, 4- M. prainii 5- M. fatua, 6- M. andamanica, 7- K. andamanica, 8- M. amygdalina, 9- Unidentified species, 10- Gymnocranthera canerica 13

14 14 Conclusions: A short and cost effective protocol could be optimized for isolation of DNA from Myristica fragrans, for screening large germplasm accessions using molecular methods A protocol was optimized for isolation of DNA from highly recalcitrant wild species of Myristica rich in polyphenols and polysacchrides Optimised conditions for amplifying DNA by PCR using ISSR, RAPD and ITS primers from the recalcitrant wild species of Myristica and also Myristica fragrans Unique markers identified and finger prints generated in each species can be used for the unequivocal identification of the individual for protection of IPR Molecular characterization of elite accessions of nutmeg (Myristica fragrans Houtt.) using RAPD, ISSR and rdna RFLP markers. An attempt was made to develop a molecular method for determining the genetic diversity in closely related accessions of large germplasm repositories. A rapid and inexpensive DNA isolation protocol was optimized and used for molecular fingerprinting of selected elite accessions of Myristica fragrans by RAPD, ISSR and rdna-rflp markers (Fig 3). Among the RAPD primers, OPB 10 and OPB 08 could detect distinctly all the accessions tested (Fig 4; Table 2). Except in A9/18 and A9/25, eighteen products were identified to be accession specific. Most of the polymorphic loci were shared by or were absent in at least two accessions. Some of the polymorphic loci were shared by all accessions. Maximum number of shared loci was observed in A9/69 (76) and least in A9/22 (26) (Table 3). The number of polymorphic bands per primer is depicted in Table 4. Dendrogram constructed using cluster analysis by UPGMA showed two major clusters, with the first forming 2 subclusters with maximum members namely A9/4, A9/18, A9/25, A4/12 and A9/150, with maximum similarity between A9/25 and A9/18. A9/4, a plagiotropic epicotyl graft outgrouped from the rest in this group. The next cluster included A9/69 and A9/86. The last cluster had two members A9/22 and A4/22

15 15 upto similarity index of 64%.The similarity coefficient ranged between 0.85 and A4/22 is a type with highest number of erect shoots and it clustered separately from the rest, however, showed 68.2% similarity with A9/22. In the I major cluster all the accessions with bold fruits and high yielders were grouped. A9/150 with large fruits and thick mace and A4/12 with round elongated fruits out grouped from the rest in this cluster. The dendrogram constructed (Fig 5) based on UPGMA using paired matrix values showed the genetic relatedness among all accessions. The similarity coefficient ranged between indicating high genetic relatedness among the accessions. All the accessions were collected from various locations within the state and hence not much of geographical variations exist. However, two accessions A9/18 and A9/20 showed the highest similarity of 85%. It is indicative that the accessions are all genetically different. The average genetic distance of 25.5% among the accessions is mainly attributed to the high degree of relatedness among them. The unique bands identified in A9/4, a very high yielding accession which is an epicotyl graft with plagiotropic shoots, A9/150, possessing very thick mace and apple shaped bold fruits and in A4/22 with unique character of high number of erect shoots may be used for their unequivocal identification (Table 5). It may also be assumed that these markers are linked to those important traits associated with the accessions. Earlier workers have also reported such unique markers linked to special characters. All the accessions could be distinguished by a single RAPD marker in eight accessions, while in A9/18 and A9/25 more than one marker was needed. The number of markers required to distinguish any group of cultivars/accessions depends on genetic variability present among the cultivars/accessions and also the size of the group. RAPD and ISSR markers are considered to be good tools for assessing genetic diversity at molecular level and has been employed in phylogenetic studies in various plant species and also has been used as distinguishing tags for species, genotypes and varieties. 75% of the bands observed in RAPD were polymorphic. This high number of polymorphic bands that distinguished all the accessions in the study suggests the advantage of RAPD for identification of genetic diversity in a closely related group of

16 16 accessions in nutmeg. Atleast 50 differentiating loci is necessary to evaluate genetic variability. More than 150 markers are necessary to obtain a good precision in analyzing genetic diversity, irrespective of the technique employed. We have analysed 99 polymorphic loci. In Gossypium an average of 12.5 polymorphic bands was obtained per primer, which was 9.9 in this study. The accessions were originally collected from different locations in Kerala and were maintained for years in the germplasm repository. In spite of this, a good deal of polymorphism was detected using RAPD markers and could distinguish between the closely related accessions. The utility of RAPD markers in estimating of genetic diversity even in the closely related races/varieties of wheat is reported. A good amount of diversity could be detected in the accessions studied and hence qualifies for inclusion in the core collection. The unique bands identified can be used for marking specific accessions, as tags for future improvement and for species authentication. Table 2. Primers that showed amplification in RAPD-PCR and their sequences Sample No. Primer Sequence 1. OPA01 CAGGCCCTTC 2. OPA05 AGGGGTCTTG 3. OPA14 TCTGTGCTGG 4. OPA17 GACCGCTTGT 5. OPC06 GAACGGACTC 6. OPB08 GTCCACACGG 7. OPB10 CTGCTGGGAC 8. OPB12 CCTTGACGCA 9. OPB15 GGAGGGTGTT 10. OPB20 GGACCCTTAC 11. OPE01 CCCAAGGTCC 12. OPE08 TCACCACGGT 13. OPE09 CTTCACCCGA

17 17 Table 3. No. of amplified products / primer Sample No. Primer Total number of bands 1. OPA OPA OPA OPA OPC OPB OPB OPB OPB OPB OPE OPE OPE09 21 Table 4. Number of polymorphic bands per primer Sample no Primer No. of polymorphic bands OPA01 5 OPA05 9 OPA14 9 OPA17 9 OPC06 10 OPB08 9 OPB10 12 OPB12 10 OPB15 6 OPB20 7 OPE OPE08 3 A9/20 13 OPE09 3 A4/12 Accessions detected A9/18,A9/22,A9/86, A9/150,A4/12,A4/22 A9/4,A9/20,A9/69, A9/86,A4/22 A9/18,A9/22,A9/25,A9/69,A9/86, A9/150,A4/12,A4/22 A9/20,A9/22,A9/25,A9/69,A9/86, A9/150,A4/12,A4/22 A9/4,A9/22,A9/86, A9/150,A4/12,A4/22 A9/4,A9/18,A9/20,A9/22,A9/25, A9/69,A9/86,A9/150,A4/12,A4/22 A9/4,A9/18,A9/20,A9/22,A9/25, A9/69,A9/86,A9/150,A4/12,A4/22 A9/4,A9/22,A9/25,A9/69,A9/86, A9/150, A4/12,A4/22 A9/25, A9/86, A9/150, A4/12, A4/22 A9/4, A9/18, A9/22, A9/69, A9/86, A9/150, A4/12, A4/22 A9/4, A9/22, A9/25, A9/86, A9/150, A4/12, A4/22

18 18 Table 5. Unique bands generated Sample No. of Primer No. bands Size (bp) 1. OPA OPA OPA OPA , , OPC , OPB , , OPB OPB , OPE OPE , OPE Fig 3. (1A). Amplification profiles of selected nutmeg accessions using RAPD (a), ISSR (1B) and rdna RFLP markers (1C) M- molecular weight marker. Lanes Accessions A9/4, A9/18, A9/20, A9/22, A9/25, A9/69, A9/86, A9/150, A4/12 and A4/22.

19 Fig 4. Amplification profiles of primers showing polymorphism in selected nutmeg accessions using RAPD Lanes Accessions A9/4, A9/18, A9/20, A9/22, A9/25, A9/69, A9/86, A9/150, A4/12 and A4/22. 19

20 20 Fig 5. Dendrogram showing the genetic relatedness of the 10 elite nutmeg accessions based on RAPD data Conclusions Ten elite accessions of nutmeg possessing high sabinene coupled with low myristicin and elimicin contents were chosen to study their genetic relationship. Out of 45 primers screened 13 that yielded good polymorphism viz. OPA 01, OPA 05, OPA 14 OPA 17, OPC 06, OPB 08, OPB 10, OPB 12, OPB15, OPB 20, OPE 01, OPE 08 and OPE 09 were selected. Total number of amplification products scored was 866 ranging from 274 to 3122 bp. Total number of polymorphic bands scored were 99 (75%) ranging between %. Single primer detected between 3-12 polymorphic bands. Maximum number of polymorphic bands was observed in OPB 10 (12). Dendrogram constructed using cluster analysis by UPGMA showed 3 major clusters with similarity coefficient ranging between 0.85 and The first group had maximum members namely A9/4, A9/18, A9/25, A4/12 and A9/150. All the accessions of this cluster had bold fruits and were high yielders. A9/150 with large fruits and thick mace and A4/12 with round elongated fruits out grouped from the rest in this cluster.

21 21 A9/4, the plagiotropic epicotyl graft outgrouped from the rest. The next cluster included A9/69 and A9/86. The last cluster had two members A9/22 and A4/22. A4/22 is a type with highest number of erect shoots and it clustered seperately from the rest, however, showed 68.2% similarity with A9/22. Unique bands were observed in all the accessions except A9/18 and A9/ products were identified as unique or accession specific. OPB 10 and OPB 08 were the only primers that detected distinctly all the accessions. Most of the polymorphic loci were shared by or were absent in at least two accessions. Some of the polymorphic loci were shared by all accessions. Maximum number of such shared loci were observed in A9/69 (76) followed by A9/20 (72). The least number was observed in A9/22 (26). All the accessions under study could be distinguished by a single RAPD marker except in A9/18 and A9/25, where more than one marker was needed. All the accessions showed variation from each other and hence may be useful to be included in a core collection. 4. Molecular characterization of wild and related genera of Myristica fragrans Good quality DND (Fig 6.) could be isolated following the protocol optimized in the lab. A total of 13 RAPD, 10 ISSR primers and two restriction enzyme digestions of 18S rdna ITS amplified products was employed for screening the different species under study. The amplification products ranged from 141 to 2791 bp in case of RAPD (Table 7). Maximum number of amplification products were obtained with primer OPB 08 (27) followed by OPB 20 (8). Average number of 16.3 bands was detected per primer (Table 8). Total number of polymorphic bands scored was 194 (99%). Total number of unique bands detected here was 52 (Table 9) with maximum in OPA 14 (9) followed by OPA 17 & OPB 20 (6). All the species could be detected by unique bands (Table 6). In case of ISSR markers the amplification product ranged from 7271 to 481 bp. Maximum number of amplification product was obtained with primer ISSR 2T1 (20) followed by ISSR 2T3 (15). Average number of 10.6 bands was detected per primer. Total number of polymorphic bands scored was 72 (75%). A number of unique bands were identified in ISSR 01 and ISSR 2T 1 (4 each). All species except M. beddomi, M. malabarica and M. prainii could be identified by unique bands. Representative gels are given in Fig 7 a,b.

22 22 In case of 18S rdna ITS fingerprinting, two enzyme combinations were employed. Fig 8 a shows the amplification of DNA using ITS primers. Polymorphism was observed among the members with both the enzymes (Table 10). Maximum number of amplification product was obtained with ECOR I (8) followed by Hind III (7) Unique bands were detected only in Gymnocranthera (Fig 8 b). The Dendrogram constructed based on UPGMA showed 2 major clusters, first grouping maximum members of Myristica sp., with Gymnocranthera isolated from the rest in the 2 cluster. The first cluster was further divided into 3 subclusters, with M. fragrans, M. prainii and Knema andamanica clustering separately (Fig 9) at a similarity index of 66%. The second subcluster contained the species viz. M. beddomi, M. malabarica (80% similarity), M. amygdalina, unidentified species, M. fatua and M. andamanica. Gymnocranthera sp., which is a related genera of Myristica showed only 58% similarity with the rest of Myristica sp. under study. M. beddomi and M. malabarica were 80% similar to each other. Knema andamanica was showing more closeness to M. fragrans and M. prainii. However, Myristica andamanica was more close to M. fatua. One of the unidentified species showed more than 70% similarity to M. fatua. Fig 6. DNA isolated from different species of Myristica and related genera 1: M. malabarica 2- M. beddomei; 3-M. prainii; 4- M. amygdalina; 5- M. andamanica; 6- Gymnocranthera sp. ; 7- Knema andamanica; 8- unidentified sp.; 9- M. fatua; 10- M. fragrans

23 23 Table 6. List of unique bands formed in RAPD and ISSR analysis Primer name No. of unique bands *Species identified (no. of bands) Size (Bp) OPA01 3 6,10,4 1988,1617,321 OPA03 2 7,2 1175,1093 OPA (2)17(2),9,4 2103,1845,1889,329,1261,793,52 3 OPA17 6 5,9,7,1,3,4 1588,1520,1037,1237,435,256 OPB (2),10,5 1715,538,1519,418 OPB ,10(2),3,5 2791,1642,335,1412, 606 OPB ,7,2,5 881,468,443,319 OPB17 4 4,3,9, ,959,907,479 OPB (2),7(2) 1257,468,679,540 OPB20 6 4,7 (3),8, ,1492,1264,301,1492,485 OPE01 2 5,7 1890,1071 OPE09 3 4,5,7 709,1909,2134 ISSR ,5,8,7 6965,3077,2639,1560 ISSR02T1 4 1, 6(3) 5939,7271,4316,3261 ISSR02T ISSR ISSR ISS R16 2 7, ,4090 ISSR ,9 1071,4530 ISSR ISSR11A *Species 1- M. fragrans, 2- M. beddomei, 3- M. malabarica, 4- M. prainii, 5- M. fatua, 6- M. andamanica, 7- Knema andamanica, 8- M. amygdalina 9- unidentified species, 10- Gymnocranthera sp

24 24 M M a b c d e f Fig 7a. Representative amplification pattern of Myristica species and related genera by ISSR. a: ISSR 01, b: ISSR 2T1, c: ISSR 2T3, d: ISSR 11A, e: ISSR 13, f :ISSR M. fragrans, 2- M. beddomei, 3- M. malabarica, 4- M. prainii, 5- M. fatua, 6- M. andamanica, 7- Knema andamanica, 8- M. amygdalina 9- unidentified species, 10- Gymnocranthera

25 25 OPB 18 OPB 08 OPB 20 OPA 14 OPA 01 OPB 10 Fig 7b. Representative amplification pattern of Myristica species and related genera by RAPD. 1- M. fragrans, 2- M. beddomei, 3- M. malabarica, 4- M. prainii, 5- M. fatua, 6- M. andamanica, 7- Knema andamanica, 8- M. amygdalina 9- unidentified species, 10- Gymnocranthera

26 26 M Fig 8 a. Amplification of DNA from ten species using 18 S rdna primers M- molecular wt marker (100 bp); : 1- M. fragrans; 2- M. beddomei; 3- M. malabarica; 4- M. prainii; 5- M. fatua; 6- M. andamanica; 7- Knema andamanica; 8- M. amygdalina; 9-unidentified sp-; 10- Gymnocranthera sp A

27 M B Fig 8b. rdna RFLP silver stained gels showing polymorphism in the 18 S rdna region. A- Hind III restriction; B- ECOR I restriction Table 7. Primers that showed amplification in RAPD-PCR and their sequences Sample No. Primer Sequence 1 OPA01 CAGGCCCTTC 2 OPA05 AGGGGTCTTG 3 OPA14 TCTGTGCTGG 4 OPA17 GACCGCTTGT 5 OPA 03 AGTCAGCCAC 6 OPB08 GTCCACACGG 7 OPB10 CTGCTGGGAC 8 OPB17 AGGGAACGAG 9 OPB15 GGAGGGTGTT 10 OPB20 GGACCCTTAC 11 OPE01 CCCAAGGTCC 12 OPB18 CCACAGCAGT 13 OPE09 CTTCACCCGA

28 28 Table 8. Monomorphic and polymorphic bands generated by RAPD and ISSR primers. Primer name No. of monomorphic bands No of polymorphic bands OPA OPA OPA OPA OPB OPB OPB OPB OPB OPB OPE OPE ISSR ISSR02T ISSR02T ISSR ISSR ISS R ISSR ISSR ISSR11A Total no of bands Table 9. List of unique bands formed in RAPD and ISSR analysis Primer name No.of *Species Size(Bp) unique bands identified (no. of bands) OPA01 3 6,10,4 1988,1617,321 OPA03 2 7,2 1175,1093 OPA (2)17(2),9,4 2103,1845,1889,329,1261,793,523 OPA17 6 5,9,7,1,3,4 1588,1520,1037,1237,435,256 OPB (2),10,5 1715,538,1519,418 OPB ,10(2),3,5 2791,1642,335,1412,606 OPB ,7,2,5 881,468,443,319 OPB17 4 4,3,9, ,959,907,479 OPB (2),7(2) 1257,468,679,540 OPB20 6 4,7 (3),8, ,1492,1264,301,1492,485 OPE01 2 5,7 1890,1071 OPE09 3 4,5,7 709,1909,2134 ISSR ,5,8,7 6965,3077,2639,1560

29 29 ISSR02T1 4 1, 6(3) 5939,7271,4316,3261 ISSR02T ISSR ISSR ISS R16 2 7, ,4090 ISSR ,9 1071,4530 ISSR ISSR11A *Species 1- M. fragrans, 2- M. beddomei, 3- M. malabarica, 4- M. prainii, 5- M. fatua, 6- M. andamanica, 7- Knema andamanica, 8- M. amygdalina 9- unidentified species, 10- Gymnocranthera sp Table 10. Polymorphism detected in 18 S rdna finger printing of wild and related genera of Myristica Primer Enzyme Wild spcies Total bands Polymorphic bands Unique bands (*species identified) Monomorphic bands ECOR (10) 2 18 S rdna HIND III 8 4 1(10) 3 (*species identified)- 10- Gymnocranthera

30 30 Fig 9. Dendrogram showing the genetic relatedness among the wild and related Myristica. C1- M. fragrans, C2- M. beddomi, C3- M. malabarica, C4- M. prainii, V5- M. fatua, V6- M. andamanica, V7- Knema andamanica, V8- M. amygdalina, V9- Unidentified sp., V10- Gymnocranthera Conclusions This is the first report on molecular characterization of Myristica using RAPD and ISSR markers DNA isolated from the third leaf in young shoots of nutmeg gives the maximum yield with CTAB buffer at ph 9. The characteristic restriction patterns generated shows DNA is amenable to PCR (RAPD, ISSR and 18S rdna primers) and applications like RFLP and Southern blotting Identified and shortlisted primers showing maximum polymorphism for marker based studies in Myristica sp. Screening of 10 wild and related genera of Myristica using 13 RAPD, 10 ISSR and two restriction enzyme digestions of 18S rdna primer was done. Amplicons produced in RAPD ranged from 141 to 2791 bp.

31 31 Maximum number of amplification product was obtained with primer OPB 08 (27) followed by OPB 20 (8). Minimum was with OPE 09 (8). Average number of 16.3 bands was detected per primer. Total number of polymorphic bands scored was 194 (99%) and monomorphic bands were two. Single primer detected between 8-27 polymorphic bands. Total number of unique bands detected was 52 with maximum in OPA 14 (9) followed by OPA 17 & OPB 20 (6). All the species could be detected by unique bands. For ISSR the amplification product ranged from 7271 to 481 bp. Maximum number of amplification products was obtained with primer ISSR 2T1 (20) followed by ISSR 2T3 (15). Average number of 10.6 bands was detected per primer. Total number of polymorphic bands scored was 72 (75%) and monomorphic bands were 24. Maximum unique bands were detected in ISSR 01 and ISSR 2T1. All species except M. beddomi, M. malabarica and M. prainii could be identified by unique bands, where more than one marker is required Polymorphism was observed among the members in 18 S rdna fingerprinting with both the enzymes. The amplification product ranged from 7271 to 481 bp. Maximum number of amplification product was obtained with Hind III (7) followed by ECOR I (08). Unique bands were detected only in Gymnocranthera with both the enzymes The Dendrogram constructed based on UPGMA showed 2 major clusters, first grouping maximum members of Myristica sp., with Gymnocranthera isolated from the rest in the 2 cluster. The first cluster was further divided into 3 subclusters, with M. fragrans, M. prainii and Knema andamanica clustering separately at a similarity index of 66%. The second subcluster contained the species viz. M. beddomi, M. malabarica (80% similarity), M. amygdalina, unidentified species, M. fatua and M. andamanica. Gymnocranthera sp., which is a related genera of Myristica showed only 58% similarity with the rest of Myristica sp. under study. M. beddomi and M. malabarica were 80% similar to each other.

32 32 Knema andamanica was showing more closeness to M. fragrans and M. prainii. However, Myristica andamanica was more close to M. fatua. One of the unidentified species showed more than 70% similarity to M. fatua. Members of the Myristica sp. could be clearly distinguished from the related genera. 5. Analysis of DNA polymorphism in clonal and seedling progenies of elite nutmeg (Myristica fragrans houtt.) by RAPD Molecular profiling was done involving clonal and seedling progenies of a dwarf, high yielding, and high quality selection of nutmeg (Myristica fragrans Houtt.) very popular for its suitability to humid tropics. The RAPD profiles were analysed for detecting molecular variations and uniqueness in progenies. Number of amplified products per primer ranged from 80 (OPC 06) to 18 (OPA 05) and their sequences are depicted (Table 11 & 12). All together 405 bands were produced. The bands ranged from 2500 bp (OPC 06) to 225 bp (OPA 14). All the individuals under study gave scorable bands. Number of polymorphic loci observed was 20 with a total of 160 making the mean number of polymorphic bands/primer as 16 (Table 13). Primers OPA 01, OPA 05 and OPA 14 gave the maximum number of polymorphic loci of three each. All the primers showed monomorphic loci, the maximum given by OPA 01 (4). Extensive amount of variation for the total and average number of bands formed by each primer within each individual was observed. All the primers taken together the maximum number of bands was found in male seedling (37) and minimum in seedling No. 14 and clone No. 14 (31 each). The analysis of UPGMA using Nei and Li (1979) index was done assuming that amplification products differing in electrophoretic mobilities are non-allelic and products with same mobilities are allelic. Reproducible results could be obtained in this study, using specific combination of primer and DNA template combination. Extreme care was taken not to alter any experimental parameters. Genetic similarity was calculated assuming that comigrating amplified bands are allelic. Homology of RAPD products has been previously demonstrated in different species. RAPD analysis was shown to identify accepted relationships between genomes of Musa and Brassica, hence the concept that comigrating bands are allelic seems to be realistic. The JSI values in the present study ranged from 0.72 to 1.00 (Fig 10.). It could be inferred that high genetic affinities were

33 33 present among the individuals due to their common parentage. Dendrogram grouped all the clones and seedlings into one cluster, with the male seedling separated from the rest. Clones No. 12, 15 and seedling No. 11 showed 100% similarity with the mother. Two of the clones viz. No. 13 and No. 16 showed 96 and 93% similarity. It is interesting to note that clones showed definite variations from the mother and clone No. 13 was very close to seedling No. 16. Clonal selections are expected to be extremely difficult to differentiate using anonymous markers. All the seedlings clustered separately from the mother and the clonal progenies; however, one of the seedlings (No. 11) showed very close relationship with clones, distinguishable only by the absence of a band of 101 bp amplifiable by OPC 06. A number of unique bands could be identified, which are useful in designating the individuals under study (Table 14.). Though clonal progenies could not be distinguished from each other, all the seedling progenies could be designated with unique bands, for specific identification. No single primer could distinguish all the progenies (Table 12). The similarity index within clones (96-100%) was considerably more than that between clones and seedlings (83-100%) and within seedlings (76-90%). As expected, clones closely resembled parent (96-100%), while seedlings showed variations (86-100%). The variability induced due to sexual crossing in nutmeg is hereby confirmed. However, lack of adequate genetic variability for many important attributes has been reported in nutmeg progenies. The study thus confirms absence of obvious relationship between the extent of morphological variations and that of changes at the DNA level already reported in other crops. It is also indicated that to conserve the unique qualities of the genetically superior nutmeg, clonal propagation is better. Nutmeg being dioecious in nature variations is expected as in case of other dioecious plants like Cannabis sativa, where high degree of polymorphism existed and plant-to-plant variation could be observed. Earlier workers suggested use of similar molecular techniques for identification in case of a confusion or misidentification of genotypes where not much of a morphological variation whatsoever exists. A high potential of RAPD markers in detecting molecular polymorphism between individuals of an accession, phenotypically pure stocks and intra varietal variations among donor plants have been demonstrated.

34 34 The male seedling showed an average similarity of 72.4% with the clones and 71.6% with seedlings and 73% with the mother. The male seedling could be characterized by presence of specific bands of 1300 and 1000 bp amplifiable by OPC 16 and OPA 05 respectively and absence of a band of 1400 and 1200 amplifiable by OPA 01 and 710 bp by OPA 14 (Fig 11). It is likely that these markers are linked to sex determining loci, though the sex determination mechanism is not yet characterized in nutmeg. In papaya a PSDM was isolated. In Myristica female specific bands were isolated by RAPD. However, extensive studies involving sex pooled DNA samples from more number of individuals and primers are needed to confirm this. For molecular markers linked to specific genes there is a need for screening large number of RAPD primers, usually more than 500. Table 11. Number of amplified products/primer Primer Total no. of bands OPA01 69 OPA03 50 OPA05 18 OPA06 24 OPA14 55 OPA17 37 OPC06 69 OPC16 23 OPE08 26 OPE09 23 Total 405

35 35 Table 12. Primers exhibiting polymorphism and their sequences Sl. No. Primers Sequence 1 OPA01 CAGGCCCTTC 2 OPA03 AGTCAGCCAC 3 OPA05 AGGGGTCTTG 4 OPA14 TCGGCGATAG 5 OPA17 TTCCGAACCC 6 OPC06 GAACGGACTC 7 OPC16 CACACTCCAG 8 OPE08 TCACCACGGT 9 OPE09 CTTCACCCGA Table 13. Number of polymorphic bands per primer Primer No. of polymorphic bands OPA01 3 OPA03 2 OPA05 3 OPA06 0 OPA14 3 OPA17 3 OPC06 2 OPC16 2 OPE08 1 OPE09 1

36 36 Table 14. List of unique bands identified in clonal and seedling progenies Band Primer Comments 1400 bp OPA 01 Present only in the male seedling 1200 bp OPA 01 Present only in the male seedling 800 bp OPA 03 Absent in clone no bp OPA 05 Present in male seedling 710 bp OPA 14 Absent in male seedling 600 bp OPA 17 Absent in seedling no bp 1090 bp 620 bp OPC 06 OPC 06 OPC 06 Absent in clone no. 16 Absent in clone no. 16 Absent in seedling no bp OPC 16 Present in male seedling 179 bp OPE 08 Present in seedling no bp OPE 09 Absent in clone no.14 Fig 10. Dendrogram showing the genetic relatedness of the progenies of elite nutmeg, IISR Viswashree. 1-5: clonal progenies; 6: Viswashree mother; 7-12: seedling progenies;

37 M M Fig 11: RAPD profiles of progenies of elite nutmeg, IISR Viswashree. A: with OPC16 and B: with OPA 05 primer. M- 500 bp ladder, Lane 1-5: clones, Lane 6: Viswashree, Lane 7-11: seedlings; Lane 12: male seedling, arrow indicates male specific band. Conclusion Good quality PCR amplifiable DNA could be isolated from nutmeg leaves following the CTAB method. DNA isolated form 3 rd leaf showed less RNA and better amplification profile in PCR. Out of the 15 random OPERON primers tested, nine showed good scorable amplification. The PCR protocol resulted in reproducible patterns of amplification products in presence of a specific combination of an accession and primer. The maximum number of bands was found in male sibling and minimum in seedling no. 15. OPA01 gave the maximum number of bands, gave 3 female specific bands amplifiable by primer OPA03. All together 357 bands were produced on an average of 3.57 bands/primer. The bands ranged from 49 bp to 472 bp. A total of 19 polymorphic bands could be observed with a mean number of 2-11 polymorphic bands/primer. Clone no. 16 could be clearly distinguished from the rest by the absence of a band of 472 bp amplifiable with primer OPC 06. Similarly seedling no. 15 and 14 could be distinguished by the absence of a band of 101 bp amplifiable by OPC 06 and another of 142 bp amplifiable by OPE 09 respectively. The JSI values in the present study ranged from 0.73 to 1.00 showed the close relatedness among the plants. At a similarity index of 0.96, three groups could be distinguished, the first comprising of all the clones, mother and a seedling progeny of

38 38 Viswashree, second comprising of all the four seedlings of Viswashree and third consisting of the sole male sibling. Three out of 4 clones showed 100% similarity with the mother plant but all the clones were formed in the same cluster. Four out of five seedlings clustered separately from the mother and the clonal progenies. The similarity index within clones was considerably more than that within seedlings. Seedlings show considerable variability from the mother and between each other at molecular level. The present study indicates that to conserve the unique qualities of the genetically superior variety of nutmeg viz. Viswashree, it may be clonally propagated than through seedling progenies. RESULTS Identification of sex specific markers in Myristica using bulked DNA analysis The DNA was isolated from five female and five male trees given in Table 15. Bulked DNA analysis was performed with DNA by mixing equal quantities of female and male DNA as two separate bulks. Twenty ISSR and 165 RAPD primers were used to identify polymorphism between the female and male bulked DNA (Fig 12 & 13). The details regarding polymorphic primers are given in Table 16. Primers that showed polymorphism between male and female bulk were OPA 07, OPD 08, OPAM 13, OPAM 16, OPAF 05 & OPAM 20. The primer OPA 07 showed one polymorphic extra band in bulked DNA from female (Fig 14-18). The primer was used for further amplification of individual female DNAs, however, the polymorphic band was detected only in one female sample ie. A911. The primer OPD 08 showed one polymorphic extra band in bulked DNA from male (Fig 16), however, was absent in one male sample i.e, A9/12. The polymorphic bands obtained are indicated by red arrows in the gel picture. Maximum number of polymorphic bands observed with primer OPAM 20 was five viz. two male specific and 3 female specific bands followed by OPAF 05 with three female specific bands and OPAM 15 three bands two male and one female was observed. Minimum was with OPAF 16 (1) and OPAM 16 (1). The primer OPAM 20 showed 1000 bp and 500 bp polymorphic band in male and 900 bp, 450 bp and 100 bp polymorphic band in female. Primer OPAM 13 showed 600 bp and 250 bp polymorphic band in male

39 39 and 800 bp polymorphic band in female. Primer OPAF 16 showed 800 bp polymorphic band in male. Primer OPAM 16 showed 500 bp polymorphic band in female. The primer OPAF 5 showed a 600 bp, 900 bp, 100 bp polymorphic band in female (Fig 17,18). The experiment was repeated twice to confirm the presence of sex specific bands. However, these bands need to be confirmed in individual male and female samples. The banding profiles of bulk DNA using ISSR primers is depicted in Fig 19. Table 15. Names of different species used in the present study Tree No. Sex A9/12 male A9/26 male A9/34 male A9/35 male A9/54 male A9/2 female A9/3 female A9/4 female A9/11 female A9/8 female Fig 12. ISSR pattern of male and female lines of Myristica fragrans generated by different primers (ISSR 02C/16, ISSR 24/16, ISSR 20T/16). M-molecular weight marker (1kb ladder) lane 1, 3 and 5-male bulk; 2, 4 and 6- female bulk.

40 40 Fig 13. RAPD pattern of male and female lines of Myristica fragrans generated by different primers (OPB17, OPE09, OPA03, OPA05, OPD01, OPA07). M-molecular weight marker(1kb ladder) lane 1,3,5,7,9 and 11-male bulk; 2,4,6,8,10and 12- female bulk. M Fig 14. Amplification of male and female bulk DNA using OPA 07 primer, arrow indicates the sex specific band in the female bulk. M: 100 bp ladder; 1, 3, 5, 7: Male bulk; 2, 4, 6, 8: Female bulk

41 41 M Fig 15. Identification of female specific bands in M. fragrans in individual DNA using OPA male; 5-9 female 11: male bulk, 12: female bulk. Arrow indicates the female specific band (800 bp) present in two out of six females and absent in all males tested. Lane 1- Molecular weight Marker 1 kb ladder, lanes 1-5 males; lanes 6-12 females M Fig 16 RAPD pattern of male and female lines of Myristica fragrans generated by primer OPD 08. M- molecular weight marker (100 bp ladder)lane 1-5- male lines; 6-10 female lines; 11- male bulk; 12- female bulk. Arrow indicate the male specific band of 800 bp

42 Fig 17 RAPD pattern of bulked DNA of male and female lines of Myristica fragrans generated by primers showing sex specific bands in red arrows 42

43 43 Fig 18. RAPD of male and female lines of M. fragrans using primers OPAM 05, OPAM11, OPAM12, OPAM13, OPAM14, OPAM15, OPAM16, OPAM17. Lanes 1, 3, 5, 7, 9- male bulk; 2, 4, 6, 8, 10- female bulk Fig 19. ISSR pattern of male and female lines of Myristica fragrans using different primers (ISSR 16/14, ISSR 20/16, ISSR 15/16. 1, 3, 5- male bulk and 2, 4, 6- female bulk