Mauro Grabiele

Exploring the Genes of Yerba Mate (Ilex paraguariensis A. St.-Hil.) by NGS and De Novo Transcriptome Assembly

Yerba mate (Ilex paraguariensis A. St.-Hil.) is an important subtropical tree crop cultivated on 326,000 ha in Argentina, Brazil and Paraguay, with a total yield production of more than 1,000,000 t. Yerba mate presents a strong limitation regarding sequence information. The NCBI GenBank lacks an EST database of yerba mate and depicts only 80 DNA sequences, mostly uncharacterized. In this scenario, in order to elucidate the yerba mate gene landscape by means of NGS, we explored and discovered a vast collection of I. paraguariensis transcripts. Total RNA from I. paraguariensis was sequenced by Illumina HiSeq-2000 obtaining 72,031,388 pair-end 100 bp sequences. High quality reads were de novo assembled into 44,907 transcripts encompassing 40 million bases with an estimated coverage of 180X. Multiple sequence analysis allowed us to predict that yerba mate contains ∼32,355 genes and 12,551 gene variants or isoforms. We identified and categorized members of more than 100 metabolic pathways. Overall, we have identified ∼1,000 putative transcription factors, genes involved in heat and oxidative stress, pathogen response, as well as disease resistance and hormone response. We have also identified, based in sequence homology searches, novel transcripts related to osmotic, drought, salinity and cold stress, senescence and early flowering. We have also pinpointed several members of the gene silencing pathway, and characterized the silencing effector Argonaute1. We predicted a diverse supply of putative microRNA precursors involved in developmental processes. We present here the first draft of the transcribed genomes of the yerba mate chloroplast and mitochondrion. The putative sequence and predicted structure of the caffeine synthase of yerba mate is presented. Moreover, we provide a collection of over 10,800 SSR accessible to the scientific community interested in yerba mate genetic improvement. This contribution broadly expands the limited knowledge of yerba mate genes, and is presented as the first genomic resource of this important crop.

Comparative cytogenetics in Cyclopogon (Orchidaceae)

A cytotaxonomical description of Cyclopogon (Spiranthinae, Orchidaceae) is carried out through a deep karyotype analysis of four species from NE Argentina. Distinctive karyotype parameters concerning the chromosomes number, morphology, size and symmetry and the genome size associate to each taxon. Cyclopogon calophyllus (2n = 2x =28; 18m + 10sm), C. congestus (2n = 2x = 32; 26m + 6sm), C. elatus (2n = 2x = 28; 18m + 10sm) and C. oliganthus (2n = 4x = 64; 40m + 24sm) possess symmetrical karyotypes (i-mean = 40.01–42.84; A1 = 0.24–032; r>2 = 0.06–0.29) and excluding C. congestus (A2 = 0.26; R = 2.62) unimodality is the rule (A2 = 0.12–0.20; R = 1.73–1.92). Diploid taxa show a terminal macrosatellite in the m pair no. 2 (large arm) and share a comparable mean chromosome length (ca. 2.75 μm) and genome size (ca. 40 μm), superior to the tetraploid C. oliganthus (ca. 2 and 32 μm, respectively). The novel data added to preceding cytological, morphological and molecular approaches involving Cyclopogon and those related taxa of Spiranthinae largely based on x = 23 support the hypothesis that the unusual 2n and the karyotype morphology of Cyclopogon is an evolutionary advance within Spiranthinae with a basic reduction to x = 14 or 16 by chromosome fusions. A polyploid-dysploid series added to dibasic hybridization explain the extant 2n diversity though a paleopolyploid series on x = 7–8 is also possible.

The 18S–25S ribosomal RNA unit of yerba mate (Ilex paraguariensis A. St.-Hil.)

We present the first report of the transcribed 18S–25S ribosomal DNA (rDNA) unit of the South American crop Ilex paraguariensis. This tree is consumed as an infusion named “mate”. Next-generation sequencing data were assembled, reference-annotated and further characterized. The assembled consensus sequence of the complete transcribed rDNA unit is 6961 nt, with a coverage of ∼18,646X and 99.3% of global sequence similarity. The transcribed 18S and 25S rDNA genes of yerba mate are the first in the genus to be characterized, combined with the 5.8S that is identical to the consensus sequence of Ilex. The ITS1 and the ITS2 display species-specific regions, useful to differentiate yerba mate from adulterant taxa of the manufactured product. A replication slippage process seems significant in the evolution of the internal transcribed spacer in Ilex. The 5-external transcribed spacer consists of SR1, which encloses a large inverted repeat/hairpin region, and SR2, which is a high homology region in the Asterids clade and embrace six distinctive inverted repeat/hairpin segments. The 3ETS presents a pyrimidine-rich block associated with transcription termination, mirror-like palindromes and repetitive segments, all networking at the secondary structure level. This information will be helpful in carrying out future studies in the molecular evolution characterization of this crop and related species of Ilex.

Cytotaxonomy of Tripogandra diuretica and Tripogandra glandulosa (Commelinaceae) from NE Argentina

Abstract Karyotype features and microsporogenesis of Tripogandra diuretica (Mart.) Handlos and Tripogandra glandulosa (Seub.) Rohw. from 15 NE Argentina accessions are analysed via classical cytogenetics, CMA/DA/DAPI and Ag-NOR. The karyotype of T. glandulosa (2n = 2x = 16; 2m + 6sm + 8st) is bimodal (A 2 = 0.37; R = 2.65) and asymmetrical (A 1 = 0.63; i = 26.06; r > 2 = 0.88) (3B-Stebbins) with a genome size of 33.28 μm; two pairs are satellited, CMA+ and NOR-actives. Another four CMA+ loci and DAPI+ universal centromeric heterochromatin occur. Microsporogenesis is normal (pollen viability short stamens = 97.8%; large = 96.7%), meiotic behaviour is regular and chromosomes pair as 8II. The karyotype of T. diuretica (2n = 8x = 64; 8m + 24sm + 32st) is bimodal (A 2 = 0.27; R = 2.54) and asymmetrical (A 1 = 0.60; i = 27.32; r > 2 = 0.88) (3B-Stebbins); additionally, two pairs are satellited. Microsporogenesis is normal (short stamens = 77.8%; large = 68.5%), except for micronuclei and low pollen viability in some accessions. Meiotic behaviour is regular with 32II, acting as a cytological diploid, however the arrangement of the haploid karyotype suggests that T. diuretica, which also exhibits a reduction of the genome size (18.58 μm), constitutes an auto-octoploid taxa. Additionally, both species share cytological features common to karyotype orthoselection.

Dataset of the first transcriptome assembly of the tree crop “yerba mate” (Ilex paraguariensis) and systematic characterization of protein coding genes

This contribution contains data associated to the research article entitled “Exploring the genes of yerba mate (Ilex paraguariensis A. St.-Hil.) by NGS and de novo transcriptome assembly” (Debat et al., 2014) [1]. By means of a bioinformatic approach involving extensive NGS data analyses, we provide a resource encompassing the full transcriptome assembly of yerba mate, the first available reference for the Ilex L. genus. This dataset (Supplementary files 1 and 2) consolidates the transcriptome-wide assembled sequences of I. paraguariensis with further comprehensive annotation of the protein coding genes of yerba mate via the integration of Arabidopsis thaliana databases. The generated data is pivotal for the characterization of agronomical relevant genes in the tree crop yerba mate -a non-model species- and related taxa in Ilex. The raw sequencing data dissected here is available at DDBJ/ENA/GenBank (NCBI Resource Coordinators, 2016) [2] Sequence Read Archive (SRA) under the accession SRP043293 and the assembled sequences have been deposited at the Transcriptome Shotgun Assembly Sequence Database (TSA) under the accession GFHV00000000.

An Integrated Physical Map of the Cultivated Hot Chili Pepper, Capsicum baccatum var. Pendulum

Capsicum baccatum var. Pendulum is the most frequently cultivated chili pepper in South America. This taxon is an outstanding source for breeding programs and genetic improvement in the genus, since its traits can be introgressed into C. annuum and related species to generate viable hybrids. In this study, the chromosome complement of C. baccatum var. Pendulum 2n = 24 was deeply characterized through a sequential combination of conventional and molecular cytogenetics approaches comprising Ag-NOR staining, heterochromatic fluorescence C-DAPI and CMA/DA/DAPI bandings and fluorescent in situ hybridization (FISH) of Capsicum-derived 5S and 18S-25S ribosomal genes and spacers probes. The attained markers were systematically integrated into a physical map which allows the identification of each individual chromosome. This is the first integrated physical map of C. baccatum var. Pendulum, which in conjunction with reported genetic linkage maps of Capsicum, would be useful for future high-density genetic mapping and whole-genome studies in C. baccatum and related taxa, as well as in breeding and genetic improvement programs in chili peppers.

Dataset of the transcribed 45S ribosomal RNA sequence of the tree crop “yerba mate”

This contribution contains data related to the research article entitled “The 18S-25S ribosomal RNA unit of yerba mate (Ilex paraguariensis A. St.-Hil.)” (Aguilera et al., 2016) [1]. Through a bioinformatic approach involving NGS data, we provide information of the transcribed 45S ribosomal RNA (rRNA) sequence of yerba mate, the first reference for the Ilex L. genus. This dataset (Supplementary file 1) comprises information regarding the assembly and annotation of this rRNA unit. The generated data is applicable for comparative analysis and evolutionary studies among Ilex and related taxa. The raw sequencing data used here is available at DDBJ/EMBL/GenBank (NCBI Resource Coordinators, 2016) [2] Sequence Read Archive (SRA) under the accession SRP043293 and the consensus 45S ribosomal RNA sequence has been deposited there under the accession GFHV00000000.

FISH-mapping of the 5S rDNA locus in chili peppers (Capsicum-Solanaceae)

We present here the physical mapping of the 5S rDNA locus in six wild and five cultivated taxa of Capsicum by means of a genus-specific FISH probe. In all taxa, a single 5S locus per haploid genome that persistently mapped onto the short arm of a unique metacentric chromosome pair at intercalar position, was found. 5S FISH signals of almost the same size and brightness intensity were observed in all the analyzed taxa. This is the first cytological characterization of the 5S in wild taxa of Capsicum by using a genus-derived probe, and the most exhaustive and comprehensive in the chili peppers up to now. The information provided here will aid the cytomolecular characterization of pepper germplasm to evaluate variability and can be instrumental to integrate physical, genetic and genomic maps already generated in the genus.