Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted ‘raw’ shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

Chloroplast Genome Evolution in Four Montane Zingiberaceae Taxa in China

Chloroplasts are critical to plant survival and adaptive evolution. The comparison of chloroplast genomes could provide insight into the adaptive evolution of closely related species. To identify potential adaptive evolution in the chloroplast genomes of four montane Zingiberaceae taxa (Cautleya, Roscoea, Rhynchanthus, and Pommereschea) that inhabit distinct habitats in the mountains of Yunnan, China, the nucleotide sequences of 13 complete chloroplast genomes, including five newly sequenced species, were characterized and compared. The five newly sequenced chloroplast genomes (162,878–163,831 bp) possessed typical quadripartite structures, which included a large single copy (LSC) region, a small single copy (SSC) region, and a pair of inverted repeat regions (IRa and IRb), and even though the structure was highly conserved among the 13 taxa, one of the rps19 genes was absent in Cautleya, possibly due to expansion of the LSC region. Positive selection of rpoA and ycf2 suggests that these montane species have experienced adaptive evolution to habitats with different sunlight intensities and that adaptation related to the chloroplast genome has played an important role in the evolution of Zingiberaceae taxa.

Complete chloroplast genomes of Rubus species (Rosaceae) and comparative analysis within the genus

Background Rubus is the largest genus of the family Rosaceae and is valued as medicinal, edible, and ornamental plants. Here, we sequenced and assembled eight chloroplast (cp) genomes of Rubus from the Dabie Mountains in Central China. Fifty-one Rubus species were comparatively analyzed for the cp genomes including the eight newly discovered genomes and forty-three previously reported in GenBank database (NCBI). Results The eight newly obtained cp genomes had the same quadripartite structure as the other cp genomes in Rubus. The length of the eight plastomes ranged from 155,546 bp to 156,321 bp with similar GC content (37.0 to 37.3%). The results indicated 133–134 genes were annotated for the Rubus plastomes, which contained 88 or 89 protein coding genes (PCGs), 37 transfer RNA genes (tRNAs), and eight ribosomal RNA genes (rRNAs). Among them, 16 (or 18) of the genes were duplicated in the IR region. Structural comparative analysis results showed that the gene content and order were relatively preserved. Nucleotide variability analysis identified nine hotspot regions for genomic divergence and multiple simple sequences repeats (SSRs), which may be used as markers for genetic diversity and phylogenetic analysis. Phylogenetic relationships were highly supported within the family Rosaceae, as evidenced by sub-clade taxa cp genome sequences. Conclusion Thus, the whole plastome may be used as a super-marker in phylogenetic studies of this genus.

Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae)

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panax vietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

Insights Into Comparative Analyses and Phylogenomic Implications of Acer (Sapindaceae) Inferred From Complete Chloroplast Genomes

Acer L. (Sapindaceae) is one of the most diverse and widespread plant genera in the Northern Hemisphere. It comprises 124–156 recognized species, with approximately half being native to Asia. Owing to its numerous morphological features and hybridization, this genus is taxonomically and phylogenetically ranked as one of the most challenging plant taxa. Here, we report the complete chloroplast genome sequences of five Acer species and compare them with those of 43 published Acer species. The chloroplast genomes were 149,103–158,458 bp in length. We conducted a sliding window analysis to find three relatively highly variable regions (psbN-rps14, rpl32-trnL, and ycf1) with a high potential for developing practical genetic markers. A total of 76–103 SSR loci were identified in 48 Acer species. The positive selection analysis of Acer species chloroplast genes showed that two genes (psaI and psbK) were positively selected, implying that light level is a selection pressure for Acer species. Using Bayes empirical Bayes methods, we also identified that 20 cp gene sites have undergone positive selection, which might result from adaptation to specific ecological niches. In phylogenetic analysis, we have reconfirmed that Acer pictum subsp. mono and A. truncatum as sister species. Our results strongly support the sister relationships between sections Platanoidea and Macrantha and between sections Trifoliata and Pentaphylla. Moreover, series Glabra and Arguta are proposed to promote to the section level. The chloroplast genomic resources provided in this study assist taxonomic and phylogenomic resolution within Acer and the Sapindaceae family.

Chloroplast phylogenomics in Camelina (Brassicaceae) reveals multiple origins of polyploid species and the maternal lineage of C. sativa

The genus Camelina (Brassicaceae) comprises 7–8 diploid, tetraploid, and hexaploid species. Of particular agricultural interest is the biofuel crop, C. sativa (gold-of-pleasure or false flax), an allohexaploid domesticated from the widespread weed, C. microcarpa. Recent cytogenetics and genomics work has uncovered the identity of the parental diploid species involved in ancient polyploidization events in Camelina. However, little is known about the maternal subgenome ancestry of contemporary polyploid species. To determine the diploid maternal contributors of polyploid Camelina lineages, we sequenced and assembled 84 Camelina chloroplast genomes for phylogenetic analysis. Divergence time estimation was used to infer the timing of polyploidization events. Chromosome counts were also determined for 82 individuals to assess ploidy and cytotypic variation. Chloroplast genomes showed minimal divergence across the genus, with no observed gene-loss or structural variation. Phylogenetic analyses revealed C. hispida as a maternal diploid parent to the allotetraploid Camelina rumelica, and C. neglecta as the closest extant diploid contributor to the allohexaploids C. microcarpa and C. sativa. The tetraploid C. rumelica appears to have evolved through multiple independent hybridization events. Divergence times for polyploid lineages closely related to C. sativa were all inferred to be very recent, at only ~65 thousand years ago. Chromosome counts confirm that there are two distinct cytotypes within C. microcarpa (2n = 38 and 2n = 40). Based on these findings and other recent research, we propose a model of Camelina subgenome relationships representing our current understanding of the hybridization and polyploidization history of this recently-diverged genus.

The complete chloroplast genome of Diarthron linifolium (Thymelaeaceae), a species found on a limestone outcrop in eastern Asia

Diarthron linifolium Turcz. is an annual herb usually found in sandy soil or limestone areas. Plants in the genus Diarthron are known to have toxic chemicals that may, however, be potentially useful as an anticancer treatment. Diarthron linifolium is a unique species among the species of the genus distributed in Korea. Here, we determine the genetic variation of D. linifolium collected in Korea with a full chloroplast genome and investigate its evolutionary status by means of a phylogenetic analysis. The chloroplast genome of Korean D. linifolium has a total length of 172,644 bp with four subregions; 86,158 bp of large single copy and 2,858 bp of small single copy (SSC) regions are separated by 41,814 bp of inverted repeat (IR) regions. We found that the SSC region of D. linifolium is considerably short but that IRs are relatively long in comparison with other chloroplast genomes. Various simple sequence repeats were identified, and our nucleotide diversity analysis suggested potential marker regions near ndhF. The phylogenetic analysis indicated that D. linifolium from Korea is a sister to the group of Daphne species.

The complete chloroplast genome of Glycyrrhiza uralensis Fisch. isolated in Korea (Fabaceae)

The chloroplast genome of Glycyrrhiza uralensis Fisch was sequenced to investigate intraspecific variations on the chloroplast genome. Its length is 127,689 bp long (34.3% GC ratio) with atypical structure of chloroplast genome, which is congruent to those of Glycyrrhiza genus. It includes 110 genes (76 protein-coding genes, four rRNAs, and 30 tRNAs). Intronic region of ndhA presented the highest nucleotide diversity based on the six G. uralenesis chloroplast genomes. A total of 150 single nucleotide polymorphisms and 10 insertion and deletion (INDEL) regions were identified from the six G. uralensis chloroplast genomes. Phylogenetic trees show that the six chloroplast genomes of G. uralensis formed the two clades, requiring additional studies to understand it.