Chloroplast genome characterization of Uncaria guianensis and Uncaria tomentosa and evolutive dynamics of the Cinchonoideae subfamily.

Uncaria species are used in traditional medicine and are considered of high therapeutic value and economic importance. This work describes the assembly and annotation of the chloroplast genomes of U. guianensis and U. tomentosa, as well as a comparative analysis. The genomes were sequenced on MiSeq Illumina, assembled with NovoPlasty, and annotated using CHLOROBOX GeSeq. Addictionaly, comparative analysis were performed with six species from NCBI databases and primers were designed in Primer3 for hypervariable regions based on the consensus sequence of 16 species of the Rubiaceae family and validated on an in-silico PCR in OpenPrimeR. The genome size of U. guianensis and U. tomentosa was 155,505 bp and 156,390 bp, respectively. Both Species have 131 genes and GC content of 37.50%. The regions rpl32-ccsA, ycf1, and ndhF-ccsA showed the three highest values of nucleotide diversity within the species of the Rubiaceae family and within the Uncaria genus, these regions were trnH-psbA, psbM-trnY, and rps16-psbK. Our results indicates that the primer of the region ndhA had an amplification success for all species tested and can be promising for usage in the Rubiaceae family. The phylogenetic analysis recovered a congruent topology to APG IV. The gene content and the chloroplast genome structure of the analyzed species are conserved and most of the genes are under negative selection. We provide the cpDNA of Neotropical Uncaria species, an important genomic resource for evolutionary studies of the group.

Complete Chloroplast Genomes of Pterodon emarginatus Vogel and Pterodon pubescens Benth: Comparative and Phylogenetic Analyses.

Background: The species Pterodon emarginatus and P. pubescens, popularly known as white sucupira or faveira, are native to the Cerrado biome and have the potential for medicinal use and reforestation. They are sister species with evolutionary proximity. Objective: Considering that the chloroplast genome exhibits a conserved structure and genes, the analysis of its sequences can contribute to the understanding of evolutionary, phylogenetic, and diversity issues. Methods: The chloroplast genomes of P. emarginatus and P. pubescens were sequenced on the Illumina MiSeq platform. The genomes were assembled based on the de novo strategy. We performed the annotation of the genes and the repetitive regions of the genomes. The nucleotide diversity and phylogenetic relationships were analyzed using the gene sequences of these species and others of the Leguminosae family, whose genomes are available in databases. Results: The complete chloroplast genome of P. emarginatus is 159,877 bp, and that of P. pubescens is 159,873 bp. The genomes of both species have circular and quadripartite structures. A total of 127 genes were predicted in both species, including 110 single-copy genes and 17 duplicated genes in the inverted regions. 141 microsatellite regions were identified in P. emarginatus and 140 in P. pubescens. The nucleotide diversity estimates of the gene regions in twenty-one species of the Leguminosae family were 0.062 in LSC, 0.086 in SSC, and 0.036 in IR. The phylogenetic analysis demonstrated the proximity between the genera Pterodon and Dipteryx, both from the clade Dipterygeae. Ten pairs of primers with potential for the development of molecular markers were designed. Conclusion: The genetic information obtained on the chloroplast genomes of P. emarginatus and P. pubescens presented here reinforces the similarity and evolutionary proximity between these species, with a similarity percentage of 99.8%.

A Pipeline for the Development of Microsatellite Markers using Next Generation Sequencing Data.

Background: Also known as Simple Sequence Repetitions (SSRs), microsatellites are profoundly informative molecular markers and powerful tools in genetics and ecology studies on plants. Objective: This research presents a workflow for developing microsatellite markers using genome skimming. Methods: The pipeline was proposed in several stages that must be performed sequentially: obtaining DNA sequences, identifying microsatellite regions, designing primers, and selecting candidate microsatellite regions to develop the markers. Our pipeline efficiency was analyzed using Illumina sequencing data from the non-model tree species Pterodon emarginatus Vog. Results: The pipeline revealed 4,382 microsatellite regions and drew 7,411 pairs of primers for P. emarginatus. However, a much larger number of microsatellite regions with the potential to develop markers were discovered from our pipeline. We selected 50 microsatellite regions with high potential for developing markers and organized 29 microsatellite regions in sets for multiplex PCR. Conclusion: The proposed pipeline is a powerful tool for fast and efficient development of microsatellite markers on a large scale in several species, especially nonmodel plant species.

Complete chloroplast genome sequence of Caryocar brasiliense Camb. (Caryocaraceae) and comparative analysis brings new insights into the plastome evolution of Malpighiales.

Caryocar brasiliense (Caryocaraceae) is a Neotropical tree species widely distributed in Brazilian Savannas. This species is very popular in central Brazil mainly by the use of its fruits in the local cuisine, and indeed it is one of the candidates, among Brazilian native plants, for fast track incorporation into cropping systems. Here we sequenced the complete chloroplast genome of C. brasiliense and used the data to access its genomic resources using high-throughput sequencing. The chloroplast exhibits a genome length of 165,793 bp and the typical angiosperm quadripartite structure with two copies of an inverted repeat sequence (IRa and IRb) of 34,902 bp each, separating a small single copy (SSC) region of 11,852 bp and a large single copy (LSC) region of 84,137 bp. The annotation analysis identified 136 genes being 87 protein-coding, eight rRNA and 37 tRNA genes. We identified 49 repetitive DNA elements and 85 microsatellites. A bayesian phylogenetic analysis helped to understand previously unresolved relationships in Malpighiales, placing Caryocaraceae as a separated group in the order, with high supported nodes. This study synthetizes valuable information for further studies allowing a better understanding of evolutionary patterns in the group and providing resources for future breeding programs.

The complete chloroplast genome of Stryphnodendron adstringens (Leguminosae - Caesalpinioideae): comparative analysis with related Mimosoid species.

Stryphnodendron adstringens is a medicinal plant belonging to the Leguminosae family, and it is commonly found in the southeastern savannas, endemic to the Cerrado biome. The goal of this study was to assemble and annotate the chloroplast genome of S. adstringens and to compare it with previously known genomes of the mimosoid clade within Leguminosae. The chloroplast genome was reconstructed using de novo and referenced-based assembly of paired-end reads generated by shotgun sequencing of total genomic DNA. The size of the S. adstringens chloroplast genome was 162,169 bp. This genome included a large single-copy (LSC) region of 91,045 bp, a small single-copy (SSC) region of 19,014 bp and a pair of inverted repeats (IRa and IRb) of 26,055 bp each. The S. adstringens chloroplast genome contains a total of 111 functional genes, including 77 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. A total of 137 SSRs and 42 repeat structures were identified in S. adstringens chloroplast genome, with the highest proportion in the LSC region. A comparison of the S. adstringens chloroplast genome with those from other mimosoid species indicated that gene content and synteny are highly conserved in the clade. The phylogenetic reconstruction using 73 conserved coding-protein genes from 19 Leguminosae species was supported to be paraphyletic. Furthermore, the noncoding and coding regions with high nucleotide diversity may supply valuable markers for molecular evolutionary and phylogenetic studies at different taxonomic levels in this group.

Cytogenetics of two hylid frogs from Brazilian Cerrado.

Cytogenetic data can be useful for taxonomic and phylogenetic studies, as well as to provide information about chromosome evolution. Therefore, it may help design conservation priorities for some threatened species, such as anurans. Herein, we describe the karyotypes of Scinax constrictus and Ololygon centralis, native endemic species from the Brazilian Cerrado. Chromosome preparations for both species were stained with Giemsa for morphological analyses and then impregnated by the Ag-NOR method for localization of the nucleolar organizer region (NOR). Both species had 24 chromosomes, as confirmed by meiotic analyses, which showed 12 bivalents. Chromosome morphologies presented the same pattern for Scinax and Ololygon compared to species already karyotyped in both genera. The NOR was interstitially located in the long arm of pair 7 in S. constrictus, whereas in O. centralis it was found near the centromere in the long arm of pair 1, thus diverging from what is commonly found for other Ololygon species. Therefore, we provide the first description of the karyotype of O. centralis and the first report of the localization of the NOR for the karyotype of both species. Our study increases the cytogenetic knowledge in species of the genera Scinax and Ololygon, and provide support for further studies on the taxonomy, ecology, and evolution of hylid anurans.

Comparative cytogenetics of tree frogs of the Dendropsophus marmoratus (Laurenti, 1768) group: conserved karyotypes and interstitial telomeric sequences.

The diploid number 2n = 30 is a presumed synapomorphy of Dendropsophus Fitzinger, 1843, although a noticeable variation in the number of biarmed/telocentric chromosomes is observed in this genus. Such a variation suggests that several chromosomal rearrangements took place after the evolutionary origin of the hypothetical ancestral 30-chromosome karyotype; however, the inferred rearrangements remain unknown. Distinct numbers of telocentric chromosomes are found in the two most cytogenetically studied species groups of Dendropsophus. In contrast, all three species of the Dendropsophus marmoratus (Laurenti, 1768) group that are already karyotyped presented five pairs of telocentric chromosomes. In this study, we analyzed cytogenetically three additional species of this group to investigate if the number of telocentric chromosomes in this group is not as variable as in other Dendropsophus groups. We described the karyotypes of Dendropsophus seniculus (Cope, 1868), Dendropsophus soaresi (Caramaschi & Jim, 1983) and Dendropsophus novaisi (Bokermann, 1968) based on Giemsa staining, C-banding, silver impregnation and in situ hybridization with telomeric probes. Dendropsophus seniculus, Dendropsophus soaresi and Dendropsophus novaisi presented five pairs of telocentric chromosomes, as did the remaining species of the group previously karyotyped. Though the species of this group show a high degree of karyotypic similarity, Dendropsophus soaresi was unique in presenting large blocks of het-ITSs (heterochromatic internal telomeric sequences) in the majority of the centromeres. Although the ITSs have been interpreted as evidence of ancestral chromosomal fusions and inversions, the het-ITSs detected in the karyotype of Dendropsophus soaresi could not be explained as direct remnants of ancestral chromosomal rearrangements because no evidence of chromosomal changes emerged from the comparison of the karyotypes of all of the species of the Dendropsophus marmoratus group.