Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia.

Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be 'the Holy Grail'; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

Impact of Genomic and Transcriptomic Resources on Apiaceae Crop Breeding Strategies.

The Apiaceae taxon is one of the most important families of flowering plants and includes thousands of species used for food, flavoring, fragrance, medical and industrial purposes. This study had the specific intent of reviewing the main genomics and transcriptomic data available for this family and their use for the constitution of new varieties. This was achieved starting from the description of the main reproductive systems and barriers, with particular reference to cytoplasmic (CMS) and nuclear (NMS) male sterility. We found that CMS and NMS systems have been discovered and successfully exploited for the development of varieties only in Foeniculum vulgare, Daucus carota, Apium graveolens and Pastinaca sativa; whereas, strategies to limit self-pollination have been poorly considered. Since the constitution of new varieties benefits from the synergistic use of marker-assisted breeding in combination with conventional breeding schemes, we also analyzed and discussed the available SNP and SSR marker datasets (20 species) and genomes (8 species). Furthermore, the RNA-seq studies aimed at elucidating key pathways in stress tolerance or biosynthesis of the metabolites of interest were limited and proportional to the economic weight of each species. Finally, by aligning 53 plastid genomes from as many species as possible, we demonstrated the precision offered by the super barcoding approach to reconstruct the phylogenetic relationships of Apiaceae species. Overall, despite the impressive size of this family, we documented an evident lack of molecular data, especially because genomic and transcriptomic resources are circumscribed to a small number of species. We believe that our contribution can help future studies aimed at developing molecular tools for boosting breeding programs in crop plants of the Apiaceae family.

[DNA super-barcoding of several medicinal species in Gentiana from Yunnan province].

Gentiana is an important but complicated group in Gentianaceae. The genus covers numerous medicinal plants which are difficult to be identified. In the present study, several medicinal species in Gentiana from Yunnan province, including G. rigescens, G.rhodantha, and G. delavayi, were sequenced using the Illumina HiSeq 2500 system. Three complete chloroplast genome sequences were obtained after assembly and annotation. According to several published genome sequences of G. crassicaulis, the DNA super-barcoding of species in Gentiana was preliminarily carried out. The results revealed that chloroplast genomes of the three species were conservative with short lengths(146 944, 148 992, and 148 796 bp, respectively). The genomes encoded 114 genes, including 78 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 2 pseudogenes. Furthermore, these medicinal species in Yunnan province were identified using DNA super-barcoding based on chloroplast genomes. The results showed that the Gentiana species could be gathered into monophyletic branches with a high support value(100%). It indicated that DNA super-barcoding possessed obvious advantages in discriminating species in complicated genera. This study is expected to provide a scientific basis for the identification, utilization, and conservation of Gentiana species.

Molecular delimitation of European leafy liverworts of the genus Calypogeia based on plastid super-barcodes.

BACKGROUND: Molecular research revealed that some of the European Calypogeia species described on the basis of morphological criteria are genetically heterogeneous and, in fact, are species complexes. DNA barcoding is already commonly used for correct identification of difficult to determine species, to disclose cryptic species, or detecting new taxa. Among liverworts, some DNA fragments, recommend as universal plant DNA barcodes, cause problems in amplification. Super-barcoding based on genomic data, makes new opportunities in a species identification. RESULTS: On the basis of 22 individuals, representing 10 Calypogeia species, plastid genome was tested as a super-barcode. It is not effective in 100%, nonetheless its success of species discrimination (95.45%) is still conspicuous. It is not excluded that the above outcome may have been upset by cryptic speciation in C. suecica, as our results indicate. Having the sequences of entire plastomes of European Calypogeia species, we also discovered that the ndhB and ndhH genes and the trnT-trnL spacer identify species in 100%. CONCLUSIONS: This study shows that even if a super-barcoding is not effective in 100%, this method does not close the door to a traditional single- or multi-locus barcoding. Moreover, it avoids many complication resulting from the need to amplify selected DNA fragments. It seems that a good solution for species discrimination is a development of so-called "specific barcodes" for a given taxonomic group, based on plastome data.

The low level of plastome differentiation observed in some lineages of Poales hinders molecular species identification.

Chloroplast genomes are a source of information successfully used in various fields of plant genetics, including molecular species identification. However, recent studies indicate an extremely low level of interspecific variability in the plastomes of some taxonomic groups of plants, including the genus Stipa L., which is a representative of the grass family. In this study we aimed to analyze the level of chloroplast genome diversity within particular genera as well as the effectiveness of identifying plant species in the Poaceae family and the other representatives of Poales order. Analysis of complete plastid genome alignments created for 96 genera comprising 793 species and 1707 specimens obtained from the GenBank database allowed defining and categorizing molecular diagnostic characters distinguishing the analyzed species from the other representatives of the genus. The results also demonstrate which species do not have any species-specific mutations, thereby they cannot be identified on the basis of differences between the complete chloroplast genomes. Our research showed a huge diversity of the analyzed species in terms of the number of molecular diagnostic characters and indicated which genera pose a particular challenge in terms of molecular species identification. The results show that a very low level of genetic diversity between plastomes is not uncommon in Poales. This is the first extensive research on super-barcoding that tests this method on a large data set and illustrates its effectiveness against the background of phylogenetic relationships.

The Increase of Simple Sequence Repeats during Diversification of Marchantiidae, An Early Land Plant Lineage, Leads to the First Known Expansion of Inverted Repeats in the Evolutionarily-Stable Structure of Liverwort Plastomes.

The chloroplast genomes of liverworts, an early land plant lineage, exhibit stable structure and gene content, however the known resources are very limited. The newly sequenced plastomes of Conocephalum, Riccia and Sphaerocarpos species revealed an increase of simple sequence repeats during the diversification of complex thalloid liverwort lineage. The presence of long TA motifs forced applying the long-read nanopore sequencing method for proper and dependable plastome assembly, since the length of dinucleotide repeats overcome the length of Illumina short reads. The accumulation of SSRs (simple sequence repeats) enabled the expansion of inverted repeats by the incorporation of rps12 and rps7 genes, which were part of large single copy (LSC) regions in the previously sequenced plastomes. The expansion of inverted repeat (IR) at the genus level is reported for the first time for non-flowering plants. Moreover, comparative analyses with remaining liverwort lineages revealed that the presence of SSR in plastomes is specific for simple thalloid species. Phylogenomic analysis resulted in trees confirming monophyly of Marchantiidae and partially congruent with previous studies, due to dataset-dependent results of Dumortiera-Reboulia relationships. Despite the lower evolutionary rate of Marchantiales plastomes, significant barcoding gap was detected, even for recently divergent holarctic Conocephalum species. The sliding window analyses revealed the presence of 18 optimal (500 bp long) barcodes that enable the molecular identification of all studied species.

DNA super-barcoding of several medicinal species in Gentiana from Yunnan province / 中国中药杂志

Gentiana is an important but complicated group in Gentianaceae. The genus covers numerous medicinal plants which are difficult to be identified. In the present study, several medicinal species in Gentiana from Yunnan province, including G. rigescens, G.rhodantha, and G. delavayi, were sequenced using the Illumina HiSeq 2500 system. Three complete chloroplast genome sequences were obtained after assembly and annotation. According to several published genome sequences of G. crassicaulis, the DNA super-barcoding of species in Gentiana was preliminarily carried out. The results revealed that chloroplast genomes of the three species were conservative with short lengths(146 944, 148 992, and 148 796 bp, respectively). The genomes encoded 114 genes, including 78 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 2 pseudogenes. Furthermore, these medicinal species in Yunnan province were identified using DNA super-barcoding based on chloroplast genomes. The results showed that the Gentiana species could be gathered into monophyletic branches with a high support value(100%). It indicated that DNA super-barcoding possessed obvious advantages in discriminating species in complicated genera. This study is expected to provide a scientific basis for the identification, utilization, and conservation of Gentiana species.