Molecular Characterization of Opium Poppy (Papaver somniferum) Germplasm

Problem statement: The medicinal value of opium poppy (2n = 22, Papaver somniferum L.) is due to presence of more than two dozen alkal oids. The genetic origin and diversity of cultivars used in the breeding collection is not fully known. To e valuate the genetic diversity of the 24 germplasm currently cultivated in the state to provide geneti c information about heterogeneity, using RAPD and ISSR markers. Approach: Isolated genomic DNA was subjected to PCR amplific ation using either random or ISSR markers. The amplified fragments wer e scored and matrix was prepared. Jaccard similarity coefficients were calculated and dendogr am was generated. Results: RAPD with 12 primers gave 46 scorable fragments out of which 32 bands we re polymorphic (69.52%) with 2.6 polymorphic band per primer, while in ISSR analysis 9 primer ga ve good amplification with DNA of 24 opium poppy germplasm. They gave 27 scorable polymorphic with 3.0 bends per primer. Dendrogram constructed revealed that the based on RAPD the ger mplasm cultivated comprised of one major group A and one minor group B, while two clusters of two germplasm each stands apart, on the basis of ISSR they fall into one major group A and one minor grou ps B, while one germplasm clusters of UOP-60 stands apart. Based on combined RAPD and ISSR analy sis these germplasm were divided into 1 major group A and 3 minor group B, C, D whereas one genot ype UOP-6 stood apart. Conclusion: These results showed that the majority of cultivated germ plasm fall in one cluster and have low genetic diversity.


INTRODUCTION
The opium poppy (2n = 22, Papaver somniferum L.) belonging to the family Papaveraceae, is an annual medicinal herb. The medicinal value of opium poppy is due to presence of numerous alkaloids out of which morphine, codeine, narcotine, thebaine and papaverine, are frequently used as pain killer, sedative, an analgesic, anti-tussive and anti spasmodic in modern medicine.
India is leading country in the production of opium poppy and is the world's largest producer of licit opium. India is the only country authorized by the United Nations single convention on narcotic drugs (1961) to produce gum opium. It is the largest producer of opium gum for the world's pharmaceutical industry. In 2002 India produced 820 Mt. of opium gum from 18447 ha. of cultivated area.
Plant breeders all over the country have developed various high yielding varieties, however most of them are yet to be characterized for their genetic diversity and gene pool. Molecular markers have been proved to be valuable tools in the characterization and evaluation of genetic diversity within and between species and populations. It has been shown that different markers might reveal different classes of variation [1,2] . It is correlated with the genome fraction surveyed by each kind of marker, their distribution throughout the genome and the extent of the DNA target which is analyzed by each specific assay [3] . RAPD markers are commonly used because they are quick and simple to obtain enabling genetic diversity analysis in several types of plant material such as natural populations, population in breeding program and germplasm collections [4] . RAPD markers are superior when simplicity and costs were considered [5] . RAPD has been used in analysis of genetic distance in different plant species [6][7][8] . ISSRs are the regions which lie within the microsatellite repeats and offer great potential to determine intergenomic diversity compared to other arbitrary primers, since they reveal variation within unique regions of the genome at several loci simultaneously. Several properties of microsatellites such a high variability among taxa, ubiquitous occurrence and high copy number in eukaryotic genome make ISSR extremely useful marker. They exhibit specificity of sequence-tagged site marker but need no sequence information for primer synthesis. ISSR usually detect a higher level of polymorphism then that detected with RFLP or RAPD analysis. In the present investigation we report genetic diversity in 24 poppy cultivars/lines from Rajasthan using RAPD and ISSR markers.

MATERIALS AND METHODS
The present investigation was conducted on leaves collected from opium poppy germplasm grown under AICRP project on opium poppy, Department of Plant Breeding and Genetics, RCA, Udaipur. Twenty four different germplasm of opium poppy from Rajasthan were taken Chemicals used in the present study were purchased from firms like BDH, E Merk, Himedia, SRL and Banglore Genei, India The genomic DNA was isolated from powdered leaf tissue using CTAB and treated with RNase to eliminate RNA. DNA concentration and purity was measured by UV-absorbance. The integrity of the isolated DNA was verified by visualization of DNA on 0.8% Agarose gel with DNA standard uncut lambda DNA.
RAPD and ISSR analysis: RAPD and ISSR analysis was done by 15 random decamer primers and 16 ISSR primers obtained from Banglore Genei Pvt. Ltd. Banglore and Sigma Co. St luis respectively. PCR reactions were performed in final volume of 25 µL containing 10 × Assay Buffer, 1 units of Taq. DNA polymerase, 250 µM each of dNTPs, 10 pmols/reaction of primer and 25 ng of template DNA. The PCR was performed in 'Thermocycler' using the following cycling parameters. After amplification, the PCR products of RAPD were loaded on 1.6% Agarose gel while in case of ISSR they were loaded on 2.5% Agarose gel (Merk) prepared in 1 × TAE buffer containing 0.5 µg mL −1 of the ethidium bromide. The amplified products were electrophoresed for 2.5-3 h at 50 V with cooling. After separation the gel was viewed under UV transilluminator and photographed with the help of gel documentation system (Alpha DG DOC).

Scoring of the RAPD and ISSR products:
In order to score and preserve banding pattern photograph of the gel was taken by a Gel Documentation System, under UV transilluminator. Bands were designated on the basis of their molecular sizes (length of polynucleotide amplified) using 100 bp DNA ladder (Hiper Himedia) as reference. The presence of each band was scored as '1' and while absence was marked as '0'. Faintly visible bands were not scored, but a major band corresponding to faint bands was considered for scoring. The matrix obtained by both RAPD and ISSR marker were analyzed by NTSY-pc software version 2.02 and dendrogram was generated by using UPGMA method.

RAPD:
Out of fifteen decamer random primer used, 12 gave amplified products. Each RAPD products was assumed to represent a single locus and data were scored as (1) and (0) for presence and absence respectively. Electrophoresis pattern of RAPD amplified product on 1.6% Agarose gel is shown in Fig. 1 with primers OPAR 11 and OPP09. Table 1 shows the polymorphic amplicon generated by different primers, Primer OPP 12 gave 3 bands in the range of 600-1000 bp with 100% polymorphism, while 3 bands were produced by OPP-16 primer within the range of 400-1000 bp and showed 100% polymorphism. Two other primer named OPAP02 and OPF17 also showed 100% polymorphism with 4 and 3 bends respectively. However 50% polymorphism were showed by two primers OPF-19 and OPA-01 with 6 and 2 bands respectively,  Total 46 fragments were amplified in 24 genotypes with 12 primers giving an average of 3.8 fragment per primer. Out of these 46 bands 32 bands were polymorphic and the level of average polymorphism was 69.56% (Table 1.) with average 2.6 polymorphic bands per primer.
The amplified fragments obtained were in the range of 200-1000 bp. The maximum numbers of amplified bands were seen in genotype UOP-5, MOP-506 and IC-19 with 43 bands with 12 primers while genotype UOP-6 and UOP-83 had only 33 bands (data not given).

ISSR:
Out of sixteen primers used 9 primers gave amplified products. Electrophoresis pattern of ISSR profile on 2.5% Agarose gel is shown in Fig. 2 with P-4 and P-1 primers. Table 2 shows the details about the polymorphic amplicon generated by different primers. Primer P-4 gave 6 bands in the range of 600 bp to 1000, 1000+1 and 1000+2 bp with 100% polymorphism, while 4 bands were produced by primer P-3 within the range of 700-1000 bp and showed 100% polymorphism. Three primer named P-1, P-8 and P-10 gave 3 polymorphic bends, while. Four primers P-5, P-11, P-13 and P-16 gave 2 polymorphic bands. Remaining 7 primers failed to amplify.    These results indicate that the majority of genotypes (sub-group A1) are close relativities.

ISSR:
Genetic similarity estimation based on ISSR banding patterns were calculated using method of Jaccard's coefficient analysis. The similarity coefficient matrix generated for the primers was subjected to algorithm UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and clusters were generated using NTSYS 2.02 pc program). The dendrogram showing relationships among various varieties was constructed using these clusters (Fig. 4). The Jaccard's similarity coefficient values ranged from 0.100 (UOP-60 and MOP-518) to 0.96 (UOP-74 and UOP-44) with an average of 0.510. The dendrogram based on ISSR analysis has generated one major group A and one minor groups B, while one germplasm clusters of UOP-60 stands apart. The group A consists of two sub groups (A1 and A2) The sub group A1 consisted of 16 genotypes viz. UO-7982, UOP-1, UOP-4, UOP-83, UOP-5, UOP-34, UOP-44, UOP-43, UO17682, MOP-50, IC-19, UOP-57, UOP-74, MOP-9, MOP-518 and NBPGR-5, whereas sub group A2 consisted of 4 genotypes viz.UOP-6, UOP-53, IC-95 and NBPGR-2. However the group within the sub group A1 consisted of 2 genotypes (UO-7982 and UOP-1) had higher within group similarity of 100% while it is 55.2% for sub group (A1). The sub group A1 and A2 joined together at the similarity level of 26.7%, whereas minor groups B joined major group at the similarity level of 34 and 70% respectively. However the average diversity estimated was very low 26.7% with a range from 7.5-44.45%. While genotype UOP-60 joined cluster at 17% similarity level. for sub group (A1). The sub group A1 and A2 joined together at the similarity level of 67.4%, whereas minor groups B joined major group at the similarity level of 60.1 and 75% respectively. The sub group C joined major group at the 62.3% with 4 genotypes (UOP-53, UOP-60, IC-95 and UOP-78) and where as group D joined major group at the similarity level of 70.8% with 2 genotypes (CHETAK and MOP-1054). However the average diversity estimated was very low 26.7% with a range from 7.5-44.45%, while genotype UOP-6 joined cluster at 62.3% similarity level.

DISCUSSION
Genetic similarity of P. somniferum genotypes estimated by a similarity matrix after RAPD analysis revealed maximum similarity between genotypes UOP-60 and IC-95. While genotypes UOP-57 with NBPGR-2 were most diverse. Fourteen genotypes fall in one cluster. ISSR marker analysis reveal that 16 genotypes fall in same cluster and that the genotypes UOP-60 and MOP-518 are most diverse while genotypes UOP-74 and UOP-44 are similar. Combined RAPD-ISSR analysis also shows a cluster of 15 genotypes indicating that these genotypes have low genetic diversity.

CONCLUSION
These results suggest that the majority of germplasm cultivated in the state fall in one cluster and have low genetic diversity.