MSc

Black pepper (Piper nigrum L.) famous as “Black Gold” and also known as “King of Spices” is one of the important agricultural commodities of commerce and trade in India since pre-historic period. The crop is the major source of income and employment for rural households in the predominantly pepper growing State of Kerala where more than 2.5 lakh farm families are involved in pepper cultivation. Karnataka, Tamil Nadu are the other major pepper producing States in the country. Kerala accounts for 80-90% of the total pepper production in the country. Idukki and Wynadu are the two major pepper producing districts in Kerala. Different cultivars/varieties are popular among the farmers and there phenotypic identity is not very distinct.

The study entitled “DNA fingerprinting of selected black pepper (Piper nigrum L.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2009-2011. The objectives of the study were to characterize the released black pepper varieties of KAU using different molecular markers - RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected.

Seven black pepper varieties (Panniyur 1 to Panniyur 7) collected from Pepper Research Station, Panniyur and maintained at CPBMB, COH were used for the study. DNA extraction was done with CTAB (Rogers and Benedich, 1994) method with slight modification. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis.
The PCR conditions were optimized for RAPD, ISSR and SSR assay. 30 RAPD, 34 ISSR and 29 SSR primers were screened with bulked DNA of black pepper varieties for amplification and those which gave reliable distinct banding pattern were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analysis were separated on 1.3 to 2 percent agarose gel and the amplification patterns recorded.
The genomic DNA from each variety was amplified with 10 each of selected RAPD and ISSR primers and 8 SSR primer pairs. The amplification pattern was scored and depicted to develop fingerprint for each variety.
The Resolving power (Rp) worked out for the different primers ranged between 7.42 to 9.42 in RAPD and 5.42 to 12.28 in ISSR analysis; indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.86 to 0.90 for RAPD analysis and it was between 0.80 and 0.89 in ISSR analysis indicating the variability among the genotypes.

Distinct bands were used to develop DNA fingerprint of black pepper varieties Panniyur 1 to Panniyur 7 through RAPD, ISSR and SSR analysis. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint of each variety with RAPD, ISSR and SSR data was also developed.
The amplification pattern observed in RAPD, ISSR and SSR analysis was scored and analyzed for quantifying the variability among the varieties. The computer package NTSYS-Pc was used for cluster analysis. Maximum variability observed was 48 percent for the variety Panniyur 4. The varieties Panniyur 1 and Panniyur 3 having the same parentage indicated 76 percent similarity. The fingerprint developed was good enough to provide varietal identity and the analysis could reveal variability/relatedness among the seven varieties.

Separate and combined fingerprints were developed for all the seven varieties through RAPD, ISSR and SSR analysis. The DNA fingerprints thus developed could be utilized for the variety registration and settling IPR issues.