Nd 2000 uv spectrophotometer

The shoots of P. kurroa plants without treatment and treated with optimum concentration of CA, CAT and CA+CAT were used for isolation of genomic DNA as per the method reported by Murray and Thompson23 (link). Total RNA was isolated by using TRIzol reagent (Ambion) as per the manufacturer’s instructions. The quantification of isolated RNA was carried out at 260 nm and 280 nm wavelengths with ND-2000 UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and quality was checked on 1% (w/v) agarose gel stained with ethidium bromide.

Total RNA of the M. canadensis samples was extracted using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. The quality and concentration of RNAs were measured using a ND-2000 UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and Agilent 2100 (Agilent, Santa Clara, CA, USA). cDNA library construction and Illumina sequencing were performed by Gene Denovo Biotechnology Co. (Guangzhou, China). Briefly, mRNA was enriched from total RNA by Oligo(dT) beads and then fragmented into short fragments. The fragmented mRNA was reverse transcribed into cDNA with random primers and second-strand cDNA was subsequently synthesized. Then the cDNA fragments were purified, end repaired, poly(A) added, and ligated to sequencing adapters. After size selection by agarose gel electrophoresis, the ligation products were PCR amplified and sequenced using Illumina HiSeq^TM 4000 (Illumina, San Diego, USA).

Total RNA was quantified at 260 and 280 nm wavelengths with the help of a ND-2000 UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Synthesis of cDNA was carried out with 5 μg of total RNA by using Verso cDNA synthesis kit (Thermo scientific, USA) as per the manufacturer’s instructions. The quantification of cDNA was performed to obtain equal concentration (100 ng) with the help of ND-2000 UV spectrophotometer. A total of 25 selected genes from glycolysis, serine biosynthesis and diterpene alkaloid biosynthesis were subjected to qRT-PCR analysis. The primers of the selected genes were designed from transcriptomic sequences of A. heterophyllum (Pal et al. 2015 (link)) by using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). The details of the primers with annealing temperatures are provided in supplementary Table 1. The expression analysis of selected genes was conducted in quadruplicates on CFX96 system (Bio-Rad Laboratories; Hercules, CA, USA) as described in Kumar et al. (2015d ). The reference genes, 26S and GAPDH were used as the standard genes in this study.