2.10 cDNA preparation and PCR-amplification of partial gene fragments
Total RNA was extracted from petals (100mg) of J. auriculatumflowers using the TRIzol® reagent (Ambion® by life technologies). cDNA was prepared from the extracted RNA (2µg) by using RevertAid TM M-MuLV reverse transcriptase (ThermoScientific) and oligo (dT)18 primer. Gene specific primers for selected volatile pathway enzymes were designed using Primer 3 (v.0.4.0) software from the transcriptome data of a closely related Jasminum species (Li et al., 2015); the gene specific primers for amplification ofJaMTS was obtained from partially cloned MTS cDNA fragment as already mentioned in Barman et al. (2020). The primers for amplification of jasmine specific PAL gene (KR869114.1) were obtained from Bera et al. (2017). The nucleotide sequences of these primers are listed in supplementary file, Table S1 .
Amplification of these genes from cDNA of J. auriculatum was performed via standard semi-quantitative PCR techniques. The primary step of denaturation was maintained at 94°C for 3 mins. This was followed by a cyclic reaction involving denaturation at 94°C for 45 s, annealing temperature (mentioned in Table S1 ) for 45 sec and extension at 72°C for 1 min. Final extension was carried out at 72°C for 10 min. All amplified products were confirmed by running in 1.5% agarose gel. The obtained amplified PCR product was eluted with QIA quick Gel extraction kit (QIAGEN, Germany), dried and sequenced at Eurofins Genomic India Pvt. Ltd. (Bangalore, India) for confirmation of gene. Gene sequences were submitted in GenBank database for obtaining accession numbers.