Hiseq4000 pe150 sequencing platform

Petals were selected from the 10 phenotypically similar plants of ‘FT’, pdm, and pdm + JA, respectively. The extraction of total RNA and cDNA synthesis were respectively conducted using an RNApure Total RNA Kit (Aidlab, Beijing, China) and a HiScript II Q Select RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Equal amounts of total RNA from three biological replicates of ‘FT’, pdm, and pdm + JA were respectively pooled for RNA-Seq library construction, which we designated as FT-1, FT-2, and FT-3; pdm-1, pdm-2, and pdm-3; and pJA-1, pJA-2, and pJA-3.
The quantity and purity of total RNA were determined using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, Carpinteria, CA, US) with RIN number > 7.0. The nine cDNA libraries were sequenced with 6 G depth using the Hiseq4000-PE150 sequencing platform (Illumina, San Diego, CA, USA) by Novogene (Beijing, China).

Whole-genome DNA was extracted from 10 g samples of fresh leaves using an E.Z.N.A.^® XXX DNA Kit. An Illumina TruSeq™ Nano DNA Sample Prep Kit was used to construct a PE library, and an 8-cycle enriched library was amplified by PCR. The target band was recovered in 2% Certified Low Range Ultra Agarose. PicoGreen nucleic acid dye was quantitatively detected with a TBS380 microfluorometer and mixed in proportion to the obtained data. A TruSeq PE Cluster Kit V3-BOT-HS was used to amplify and generate DNA clusters by bridging PCR with the cBot System. Finally, the DNA clusters were sequenced on the Illumina HiSeq 4000-PE150 sequencing platform to produce the original sequences (raw read length of 150 bp). The original sequences were subjected to quality control, whereby adapter sequences and bases containing non-AGCT at the 5′ end were removed, reads with sequencing quality values less than Q20 were trimmed, reads with N proportions more than or equal to 10% were removed, and joint sequences and small segments with lengths less than 75 bp were discarded after pruning. As a result, high-quality read sequences (clean reads) were obtained. The NT library was randomly selected to detect whether the sequencing results were contaminated.

An Illumina paired-end library was constructed by the NEBNext® Ultra™ II DNA Library Preparation Kit (New England Biolabs (Beijing) Ltd., China) and sequenced on the Illumina HiSeq 4000 PE150 sequencing platform. Approximately 17.5 Gb of raw data was generated, and the raw sequence reads were filtered for primer/adaptor sequences and low-quality reads with the NGS QC Tool Kit [22 (link)]. Sequencing reads were assembled using SPAdes 3.6.1 software [23 (link)] with the parameter Kmer=95, and 198,659 contigs were finally obtained.
MISA software [24 (link), 25 (link)] was used to identify unique reads containing microsatellite repeats. The search was performed for a minimum repeat number of 5, 4, 3, 3 and 3 for di-, tri-, tetra-, penta-, and hexa-nucleotides, respectively. Primers were designed on the basis of flanking sequences of SSR microsatellite loci by using Primer 3. The parameters of primer design were set as follows: the primer size was between 18 and 25 bp with an optimal size of 22 bp, the annealing temperature was between 55 and 65 °C with the optimal temperature of 60 °C, the PCR product size was between 80 and 300 bp, and default values were selected for other settings.