MSc

The study entitled “Expression profiling of auxin responsive genes during inflorescence development in black pepper (Piper nigrum L.)” was conducted during 2017-2019 at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram. The objective was to study the expression of auxin responsive genes such as Barren stalk fastigiate1 (BAF1), Barren inflorescence2 (BIF2) and Ramosa2 (RA2) during inflorescence development in black pepper (Piper nigrum L.) by Reverse Transcription quantitative PCR (RT-qPCR).
Auxin signalling plays an important role in positioning of axillary meristems and determining the inflorescence architecture. The auxin responsive genes Barren stalk fastigiate1 (BAF1), Barren inflorescence2 (BIF2) and Ramosa2 (RA2) are key genes governing inflorescence architecture.
Spike samples of three cultivars of black pepper viz., ‘Thekken’, ‘Karimunda’ and ‘Panniyur-1’ were used for the study. Samples were collected at three different developmental stages viz., stage 1 (1-2 cm), stage 2 (6-8 cm) and stage 3 (9-12 cm) from two different plants of each cultivar. Genomic DNA and RNA were extracted by modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and TRIzol method respectively.
Primers were designed for Barren stalk fastigiate1 (BAF1), Barren inflorescence2 (BIF2) and Ramosa2 (RA2) using “Primer Express” software and absence of secondary structure at the primer binding site was confirmed by “mfold” web server. Specificity of the designed gene specific primers was checked by PCR using genomic DNA. All the primers produced amplicons of expected size in PCR, indicating specificity of the designed primers. Amplification efficiency of the designed primers was determined by standard curve analysis and “Lin Reg” software. All the primers exhibited cent per cent amplification efficiency.
RNA isolated from the spike samples was converted to cDNA and the quality was confirmed by PCR and agarose gel electrophoresis. cDNA was used for RT-qPCR using SYBR® Green dye-based assay. Thermal conditions for RT-qPCR were 95°C for 2 min followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec and 72°C for 45 sec. RT-qPCR for each gene was performed with three technical replicates for each sample and Cq values were obtained.
Gene expression analysis was performed using “qbase plus” software using Actin as the reference gene and the amplification factor as two for all the genes. Differential expression of the three genes was noticed in all the stages of inflorescence development in the three cultivars. The expression of BIF2 was maximum at stage 2 of spike development in ‘Karimunda’ and ‘Panniyur-1’, whereas in ‘Thekken’ it was at stage 3. The expression of BAF1 and RA2 was downregulated at stage 3 of ‘Panniyur-1’ and ‘Karimunda’ and upregulated in stage 3 of ‘Thekken’.
To conclude, auxin responsive genes BAF1, BIF2 and RA2 showed higher levels of expression in the first two stages of ‘Karimunda’ and ‘Panniyur-1’ whereas in stage 3 the levels were decreased. However, initial stages of spike development in ‘Thekken’ had a lower level of gene expression and the third stage showed the highest level of expression. Expression levels at stage 3 in ‘Thekken’ was considerably higher compared to the other two cultivars. Delayed induction of auxin responsive genes in ‘Thekken’ indicate a probable role of auxin signalling in inflorescence branching. Further studies involving other auxin responsive genes are needed for confirming the role of auxin signalling during inflorescence development in black pepper.