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.