*3.2. Library Construction, Sequencing and Assembly*

Input material for the DNA sample preparations was derived (or taken) from a total amount of 1.0 μg DNA. The NEBNext DNA Library Prep Kit was used to generate sequence libraries according to the manufacturer's recommendation; indices were also added to each sample. Genomic DNA was randomly fragmented by shearing to a size of 350 bases in length. The ends of randomly fragmented DNA were repaired and A-tailed, adapters were ligated with NEBNext for Illumina sequencing, then the PCR improved by P5 and indexed P7 oligo sequences. The AMPure XP system was used to purify the PCR products; subsequent findings were analyzed by the Agilent 2100 Bioanalyzer for size distribution and later quantified using real-time PCR. After pooling, the qualified libraries were fed into an Illumina HiSeq 2500 system (350 bp paired ends reads); this was based on its effective concentration and expected data volume. The raw reads (19,844,190 bp, 19,053,503 bp, 19,440,639 bp and 19,929,468 bp for *C. farinosa, C. glandulosa, M. crassifolia* and *M. oblongifolia*, respectively) were cleaned reads (5 Gb) to remove low-quality sequences and adaptors; they were then filtered for PCR duplicates using PRINSEQlite v0.20.4 [71]. The clean raw reads were subjected to de novo assembly from the whole genome sequences using NOVOPlasty 2.7.2 [72] with kmer (K-mer = 31–33). The *trnH-psbA* of *Cadaba farinosa* (KR735837.1) was used as a seed and the complete plastome of *Arabidopsis thaliana* (KX551970.1) was used as a reference for the assembly of the *Cadaba farinosa* cp genome. The assembled cp genome of *Cadaba farinosa* was used as seed and reference for the assembly of the *Cadaba glandulosa* plastome. For *Maerua crassifolia, the rpoC1* gene of *M. crassifolia* (JQ845894.1) was used as seed and the complete cp genome of *C. farinosa* was used as reference. The assembled cp genome of *M. crassifolia* was used as seed and reference for the assembly of *M. oblongifolia*. Finally, each species generated one contig that contained the complete chloroplast genome sequence.
