Myrteae species from Brazil (
Salvador et al., 2011;
Souza Moreira et al., 2019). Thus, due to its plethora of roles, a growing interest has been addressed to this group as a model for evolutionary, ecological and applied studies. For this study, leaves from
Eugenia brasiliensis, Eugenia pyriformis, Eugenia nitida, Myrcianthes pungens, Plinia edulis and
Psidium cattleianum trees were from a private
in vivo collection in Gravataí, RS, Brazil (latitude (S): 29°51’52"; longitude (W): 50°53’53") and used to isolate chloroplasts by the modified high salt method, followed by cpDNA extraction with the CTAB method (
Vieira et al., 2014). DNA quality was evaluated by electrophoresis in a 1% agarose gel, and DNA quantity was determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For each species, one genomic paired-end library of 150 nt length was generated using an Illumina HiSeq2000 platform (Macrogen). The bases with quality below Q30 and adapter contamination were trimmed using Trim Galore!0.4.2, with a 50 bases minimum allowed. Paired-end sequence reads were filtered against 28 Myrtaceae plastomes (Table S1) using BWA software (
Li and Durbin, 2009) with two mismatches allowed. Mapped reads were used for the
de novo assembly with ABySS software (
Jackman et al., 2017). The plastome scaffolds were orientated by MUMmer (
Delcher et al., 2003) using
Eugenia uniflora (NC_026450.1),
Plinia trunciflora (NC_034801.1) or
Psidium guajava (NC_033355.1) as reference genomes for species of the same or closer genus. Genes were annotated using GeSeq (
Tillich et al., 2017) by BLAST searches with 80% similarity. Circular plastome maps were drawn using the OGDRAW web toolkit (
Lohse et al., 2013). IRa and IRb boundaries were analyzed using IRScope (
Amiryousefi et al., 2018). For each species, local mVISTA (
Frazer et al., 2004) was used to pairwise align plastomes with their respective reference. An overall genome comparison was performed with BLAST Ring Image Generator (BRIG) (
Alikhan et al., 2011). Krait v0.11.4 (
Du et al., 2018) was used to search and annotate perfect SSRs using the genomes and their annotated GFF3 file. The parameters for minimum repeat numbers were 8, 4, 3, 3, 3, 3 for mono-, di-, tri-, tetra-, penta- and hexanucleotide SSRs, respectively. DNA sequencing libraries were produced for each species, and these comprised 34.1-48.7 M raw Illumina paired-end reads (summarized in Table S2). The percentage of removed reads due to trimming ranged from 1.17-1.45%. The number of filtered reads was 1.1-2.2 M reads. The obtained reads were
de novo assembled into scaffolds that completely covered each plastome, without any gaps. The number of assembled scaffolds obtained ranged from four to eight. The minimum coverage ranged from 13 to 46 reads and the maximum coverage, 1,508-3,620 reads. The complete sequences were submitted to GenBank at accession numbers MN095407 to MN095411 and MN095413 (Table 1). The complete plastomes of six Myrteae have a narrow size range, from 157,683 bp in
E. nitida to 159,631 bp in
P. edulis, similar to the size of Myrtaceae species plastomes (
Eguiluz et al., 2017 a,
b;
Machado et al., 2017). Figures S1-S6 present the genome maps for each species. Four well-defined regions are present in all newly assembled genomes. Inverted regions (IR) ranged from ~26.3 to 26.4 kbp and had the smallest size variation, up to 78 bp. Short single copy (SSC) sections have ~18.2-18.5 kbp, while long single copy (LSC) sections have ~86.4-88.2 kbp (Table 1). Protein coding sequences comprise ~50% of the genome, rRNAs and tRNAs comprise ~7%, and non-coding regions, such as introns, pseudogenes and intergenic spacers correspond to ~43% (Table 1). Genome structure analysis showed a high degree of synteny among evaluated species (Figure 1). Genomes contained 129 genes in total, corresponding to 78 single-copy protein-coding genes, 30 transfer RNA (tRNA) genes, four ribosomal genes (rRNA) and one pseudogene (
ycf1) (Figure 1, Table 1). In general, genomic features, such as size, structure, and gene abundance are similar to previously described Myrtaceae species (
Eguiluz et al., 2017a,b). Despite the similarity in genomic features, the mVISTA comparison against each respective reference genome showed that some regions display lower similarity (Figure S7 – S9). Non-coding regions, particularly the intergenic, had lower conservation and, therefore, more variation, such as
psbI-trnS, trnT-psbD, trnS-psbZ-trnG, accD-psaI, and
ndhF-rp132 in
Eugenia and
Myrcianthes; trnS-trnR, atpF-atpH, trnT-trnL, and
rpl32-trnL in
Plinia; and most intergenic regions of LSC in
Psidium. Regarding protein-coding genes, we observed a conservation decrease in
accD and
ccsA in
Eugenia species and
M. pungens; and
rpoC2 in
Plinia and
Psidium. Protein-coding genes
matK, ndhF and
ycf1, showed more nucleotide diversity (4.6 to 6.1%) in all analyzed species (Figure S7 – S10). This diversity corroborates previous studies based on plastidial genes and non-coding regions with contrasting substitution rates (
Thornhill et al., 2015;
Machado et al., 2017). Some structural changes in the IRa and IRb boundaries were found for the evaluated species (Figure 2). Within the IRb-LSC boundaries, the boundaries of the
rps19 gene were located on the left side. In the IRb region, except for
M. pungens, the IRb-LSC boundary was embedded in
rps19 and had a length of three bp in
Eugenia (Figure 2A), ~30 bp in
Plinia (Figure 2B), and 31 bp in
Psidium, contained in the IRb (Figure 2C). The IRb-SSC boundaries were embedded in the
ycf1 pseudogene, ranging from one to eight bp in
Eugenia/Myrcianthes, one and two bp in
Plinia, and only one bp in
Psidium species. The
ndhF gene was located on the right side of the IRb-SSC at a distance from the boundary of 10 bp in
E. uniflora, 36 to 121 bp in other
Eugenia, 72 bp in
M. pungens, 109 bp and 120 bp in
Plinia, and 111 bp and 124 bp in
Psidium. The SSC-IRa boundary was embedded in
ycf1, with a length of 1047 to 1080 bp in
Eugenia/Myrcianthes, 1080 and 1011 bp in
Plinia, and 1071 and 1079 bp in
Psidium in the IRa region. The
trnH-GUG gene was located on the right side of the IRa-LSC boundary ranging from 11 to 52 bp in
Eugenia/Myrcianthes, from 3 to 10 bp in
Plinia and 10 to 14 bp in
Psidium. The contraction and expansion of IR regions are measurable events of plastome evolution. These results demonstrate a genus-specific IR conservation, which can be considered one of the reasons for genome size variation among species. In this work, we present, for the first time, a characterization of IR boundaries from species of the same genus in Myrteae because they compared different genera, previous studies could not report a significant variation in IRb-SSC border within Myrteae species (
Eguiluz et al., 2017a,b;
Machado et al., 2017). All plastomes presented a similar number of SSRs. In total, over 315 SSRs were identified for each species (Table 2). The mononucleotide SSRs of A/T were the most frequent, varying in number from 85/98 in
E. brasiliensis/E. pyriformis to 93/103 in
P. edulis/P. cattleianum (Table S3). This AT richness was already demonstrated in previous studies and reflects the lower GC content in these plastid genomes (
Eguiluz et al., 2017a,b). The secondmost common were the trinucleotide SSRs, ranging in number from 61 in
E. nitida to 71 in
P. edulis. In addition, the number of SSRs located in different regions were similar: in intergenic regions ranging from 171 in
E. nitida to 185 in
P. edulis; in genes, ranging from 96 in
E. brasiliensis, E. pyriformis, M. pungens, and
P. cattleianum, and 98 in
E. nitida and
P. edulis; and in introns, ranging from 43 in
E. brasiliensis to 47 in
E. nitida, P. edulis, and
P. cattleianum (Table 2). Hexanucleotide SSRs could not be found in the genomes. All found SRRs are listed in Table S4. These SSR results provide more information on molecular markers that could be used to evaluate intra- and interspecific diversity. This work provides reference genomes for six Neotropical Myrtaceae species, increasing the genetic information available for the Myrteae tribe, and allowing the improvement of taxa sampling in further investigations into Myrtaceae evolution.