MSc

The project entitled “Analysis of differential expression of genes determining inflorescence architecture in black pepper (Piper nigrum L.) type ‘Thekken’” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2014-2016. The objective of the study was to detect the presence and differential expression of RAMOSA family genes (RA1, RA2 and RA3) that determine the inflorescence architecture and to analyse their influence on the branching trait in black pepper (Piper nigrum L.) type ‘Thekken’.
Samples used in the study viz., leaves and spikes of black pepper type ‘Thekken’ and the control non branching variety ‘Karimunda’ were collected from farmer`s field. Genomic DNA was extracted from the leaf samples and RNA was extracted from the spikes at three different stages of development (Stage I - 1 cm; Stage II - 4 cm and Stage III - 9 cm). Methods used for nucleic acid extractions yielded good quality DNA and RNA.
Degenerate primers were designed for RA1, RA2 and RA3 genes using Primer3 and Oligocalc tools and these primers were used for screening at the genome and transcriptome level by PCR and RT-PCR respectively. The amplicons obtained were resolved on agarose gel. At genomic level, a band of size 450 bp was obtained for RA2 primers, whereas RA1 primers produced four bands (600 bp, 550 bp, 400 bp and 200 bp) and RA3 primers produced two bands (650 bp and 450 bp). There was no difference in the banding profile in ‘Thekken’ and ‘Karimunda’. The RA2 specific band (450 bp) obtained in ‘Thekken’ was sequenced and showed similarity with NADH dehydrogenase subunit 2 (nad2) gene of Parinari campestris.
On screening the cDNA, the primers designed for RA1 and RA2 genes showed no amplification in both ‘Thekken’ and ‘Karimunda’. However, the primers designed for RA3 gene showed differential expression in ‘Thekken’ and ‘Karimunda’. A band of size 450 bp was obtained in stage II of the spike of ‘Thekken’, whereas no amplification was obtained in the ‘Karimunda’ variety. The amplicon obtained using RA3 primer was cloned and sequenced.
Analysis of the RA3 specific sequence using tBLASTx showed best match with fragment of chromosome 7 of Cucumis melo and BLASTn analysis showed similarity to uncharacterized mRNA sequence from Brassica napus. Clustal Omega analysis showed 39.21 and 40.94 percent identity with the reported sequences of RA3 of Zea mays and Vitis vinifera while these two sequences among themselves showed 61.30 percent identity. Conserved domain database (CDD) search revealed the presence of an integrase core domain in this sequence.
All the three sequences obtained with RA2 and RA3 primers have been deposited in the NCBI database as ‘Floral architecture related sequence isolated from branching type black pepper’ (Accession numbers: KX518738, KX518739 and KX518740).
The present study is the first report of the presence of an integrase core domain in the genome of black pepper. Differential amplification of cDNA of stage II from ‘Thekken’ and ‘Karimunda’ with RA3 primers suggests that altered expression of the region under study may play a role in the induction of spike branching in ‘Thekken’. The presence of the integrase core domain also suggests a possible role of retroviral integration in differential expression.