MSc

The study entitled “Identification of molecular marker associated with spike branching trait in black pepper (Piper nigrum L.)” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2011-2013. The objective of the study was to screen the candidate genes regulating inflorescence branching and to identify the molecular markers associated with spike branching trait in black pepper type ‘Thekken’ which shows a profuse branching in majority of the spikes (60-80 branches per spike) leading to higher berry yield. Genetic knowledge on this economically relevant trait could be useful in crop improvement programmes.
Spikes of Thekken and Karimunda variety (as control) were collected from the farmer’s plot in Idukki and investigated at the molecular, protein and microscopic level.
In molecular analysis, the presence of candidate genes known to regulate inflorescence branching, viz. BP, RA2, PIN1 and LOG1 were screened at the genome and transcriptome level by using degenerate primers. DNA was isolated using CTAB method with modifications such as increasing the concentrations of CTAB, NaCl and EDTA and including PVP and β-mercaptoethanol in the extraction buffer.
There was difference in the amplicons between Thekken type and Karimunda variety at the genomic level. Additional amplicons were seen in Karimunda variety for RA2 (200 bp), PIN1 (900 bp) and LOG1 (300 bp) gene primers. Amplicons of approx. 500 bp, 1800 bp and 500 bp were seen in both varieties with primers for RA2, PIN1 and LOG1 respectively. BP primers gave amplification (600 bp) only in Karimunda.
Good quality RNA was isolated using Trizol reagent. However, RT-PCR did not give any amplification in both Thekken and Karimunda samples.
Molecular profiles using twelve RAPD primers and two SSR primers showed no polymorphism between branched and non-branched spikes of Thekken. However, 22.8% polymorphism was exhibited between Thekken and Karimunda by RAPD.
In protein analysis using SDS-PAGE in branched and non-branched spikes of Thekken, seven prominent bands were observed, and all were monomorphic. At the microscopic level, prominent bract was a notable feature in the spikes of Thekken.
In the present study, a protocol for isolation of genomic DNA from the mature spikes of black pepper could be standardized. The study reveals that there is no difference between branched and non branched spikes of Thekken type, indicating lesser chances of Thekken being a chimera. Difference in the amplified products of PIN1 and LOG1 in Thekken and Karimunda suggest a difference in phytohormone levels (auxin and cytokinin respectively). Similarly, absence of amplification of BP in Thekken type and difference in the amplified band of RA2 in Thekken and Karimunda suggest a possible role of transcription factors in spike branching in Thekken.