Santelmo Vasconcelos

Heterochromatin and rDNA 5S and 45S sites as reliable cytogenetic markers for castor bean (Ricinus communis, Euphorbiaceae)

The increasing need for renewable energy resources has led to higher demands for biofuel, a scenario where the castor bean (Ricinus communis L.) seed oil represents a promising source of raw material. Despite that, information regarding the genome organization of R. communis is still scarce, impairing the application of modern biotechnological and breeding procedures. The present work brings the first evaluation of the mitotic chromosomes of this species, including 10 potentially interesting accessions for cultivation in semi-arid environments aiming at the biofuel production. The approach included standard staining, fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with rDNA 5S and 45S, as well as silver impregnation. All accessions were diploid with 2n=2x=20, displaying mainly metacentric chromosomes, with CMA-positive bands (GC-rich) in all pairs of the complement. After silver impregnation, one to 14 nucleoli were observed, while the FISH with rDNA 45S revealed two large sites and a variety of minor dots, and the DNAr 5S hybridized in a single pair. The observed features were discussed and compared with literature data regarding pachytene bivalents.

Molecular Markers to Access Genetic Diversity of Castor Bean: Current Status and Prospects for Breeding Purposes

The spurge family (Euphorbiaceae) is one of the most diverse and numerous clades of the angiosperms, including several species of great economic importance as rubber tree (Hevea brasiliensis), cassava (Manihot esculenta), and some oil seed crops, as candlenut (Aleurites moluccana), physic nut (Jatropha curcas) and castor bean (Ricinus communis). Castor bean, the single member of the African genus Ricinus (subfamily Acalyphoideae), presents a wide variation regarding vegetative traits such as leaf and stem colors, number and size of leaf lobes and presence of wax covering the stem (Popova & Moshkin, 1986; Savy-Filho, 2005; Webster, 1994; see Fig. 1). Depending on the environmental conditions, even the vegetative habit may vary, although it is more likely in a shrubby form (Webster, 1994). However, the most conspicuous variability is related to reproductive characters, as color shape and size of seeds, number of flowers per raceme, peduncle length and fruit dehiscence (Figs. 1 and 2) as described by Popova & Moshkin (1986).

Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae)

Endive (Cichorium endivia L.) and chicory (C. intybus L.) both have 2n = 18, but until now, there has been no detailed karyomorphological characterization. The present work evaluated five accessions of each species using FISH with rDNA probes and fluorochrome staining with CMA and DAPI. Both species presented distinct banding patterns after fluorochrome staining: while endive had proximal CMA++/DAPI− bands in the short arms of pairs 1, 2 and 3, chicory had proximal CMA-positive bands in chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively), associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA). The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group.

ISSR Primer Selection for Genetic Variability Analyses with Jaborandi (Pilocarpus microphyllus Stapf ex Wardlew., Rutaceae)

Pilocarpus microphyllus Stapf ex Wardlew. (jaborandi) is native to the North and Northeast regions of Brazil. Intensive exploitation for the extraction of pilocarpine, a valuable alkaloid for the pharmaceutical industry, has killed off or brought about a loss of vigor of naturally occurring plant populations. As a result, the species is officially listed as an endangered species of the Brazilian flora. Genetic diversity is required for the adaptation of populations to environmental changes, and maintaining it is a central objective for biological conservation. ISSRs are dominant markers widely used in genetic diversity studies of endangered species, allowing for the identification of genotypes and cultivars, as well as helping in phylogenetic studies based on DNA fingerprinting. This study presents an ISSR primer selection for genetic structure analyses of natural populations and cultivated collections of P. microphyllus.

Genomic in situ Hybridization in Triticeae: A Methodological Approach

In several plant groups, especially those with polyploid complexes as Triticum (the wheat genus, Poaceae), related species can be used as important sources of genes. In the tribe Triti‐ ceae as a whole, which comprises other important cereals as barley (Hordeum vulgare) and rye (Secale cereale), there are high rates of successful interspecific hybridization [1-2]. Due to the ease in obtaining these hybrids, plus the high amount of available information on the genomes of the species, the interspecific hybrids are potentially useful for the genetic im‐ provement of these crops [3-4]. Thus, the hybrids and their derivatives from breeding pro‐ grams can be analyzed by means of different approaches, aiming the full knowledge on the phenotypic constitution of the plant material for its subsequent utilization.

Recognition of the genus Thaumatophyllum Schott − formerly Philodendron subg. Meconostigma (Araceae) − based on molecular and morphological evidence

Abstract Philodendron subgenus Meconostigma has been a well-circumscribed group since 1829. Members of this group are easily distinguished by diagnostic morphological characters as well as by a distinct ecology and geographical distribution. Based on molecular, morphological and cytological evidence, we propose the recognition of P. subg. Meconostigma as a distinct genus, Thaumatophyllum Schott. We also present the necessary new combinations, an emended key and some nomenclatural and taxonomic corrections regarding 21 names of Thaumatophyllum.

Natural history of the narrow endemics Ipomoea cavalcantei and I . marabaensis from Amazon Canga savannahs

Amazon comprises a vast variety of ecosystems, including savannah-like Canga barrens that evolved on iron-lateritic rock plateaus of the Carajás Mountain range. Individual Cangas are enclosed by the rain forest, indicating insular isolation that enables speciation and plant community differentiation. To establish a framework for the research on natural history and conservation management of endemic Canga species, seven chloroplast DNA loci and an ITS2 nuclear DNA locus were used to study natural molecular variation of the red flowered Ipomoea cavalcantei and the lilac flowered I. marabaensis. Partitioning of the nuclear and chloroplast gene alleles strongly suggested that the species share the most recent common ancestor, pointing a new independent event of the red flower origin in the genus. Chloroplast gene allele analysis showed strong genetic differentiation between Canga populations, implying a limited role of seed dispersal in exchange of individuals between Cangas. Closed haplotype network topology indicated a requirement for the paternal inheritance in generation of cytoplasmic genetic variation. Tenfold higher nucleotide diversity in the nuclear ITS2 sequences distinguished I. cavalcantei from I. marabaensis, implying a different pace of evolutionary changes. Thus, Canga ecosystems offer powerful venues for the study of speciation, multitrait adaptation and the origins of genetic variation.

Development of 15 SSR polymorphic markers for the endangered Ameroglossum pernambucense Eb. Fischer, S. Vogel & A. V. Lopes (Scrophulariaceae), and cross-transferability in congeneric taxa

Ameroglossum (Scrophulariaceae) is an endemic genus from northeastern Brazil patchily distributed in granitic rock outcrops (inselbergs) with two species described: the endangered A. pernambucense Eb. Fischer, S. Vogel & A. V. Lopes and the narrow endemic A. manoel-felixii L. P. Felix & E. M. Almeida. We developed 15 polymorphic nuclear microsatellite markers for A. pernambucense using pyrosequencing and successfully cross-amplified 14 of these loci in A. manoel-felixii and a taxonomically uncertain taxon (a putative Ameroglossum hybrid). The number of alleles per loci ranged from 2 to 6, 2 to 5 and 2 to 6 in the examined populations of A. pernambucense, A. manoel-felixii and the putative hybrid, respectively. No significant linkage disequilibrium was detected, and significant deviation from Hardy–Weinberg equilibrium was found in some loci, likely reflecting inbreeding and population structure across inselbergs. We discuss the use of these SSR markers in conservation and microevolutionary studies in Ameroglossum, as well as in the investigation of the importance of pollen-mediated gene flow in keeping genetic diversity in rare species with patchily distributed populations using progeny arrays.

Unraveling the karyotype structure of the spurges Euphorbia hirta Linnaeus, 1753 and E. hyssopifolia Linnaeus, 1753 (Euphorbiaceae) using genome size estimation and heterochromatin differentiation

Abstract Euphorbia Linnaeus, 1753 (Euphorbiaceae) is one of the most diverse and complex genera among the angiosperms, showing a huge diversity in morphologic traits and ecologic patterns. In order to improve the knowledge of the karyotype organization of Euphorbia hirta (2n = 18) and Euphorbia hyssopifolia (2n = 12), cytogenetic studies were performed by means of conventional staining with Giemsa, genome size estimations with flow cytometry, heterochromatin differentiation with chromomycin A3 (CMA) and 4’,6-diamidino-2-phenylindole (DAPI) and Giemsa C-banding, fluorescent in situ hybridization (FISH) with 45S and 5S rDNA probes, and impregnation with silver nitrate (AgNO3). Our results revealed small metacentric chromosomes, CMA+/DAPI0 heterochromatin in the pericentromeric regions of all chromosomes and CMA+/DAPI− in the distal part of chromosome arms carriers of nucleolar organizing regions (NORs). The DNA content measurements revealed small genomes for both species: Euphorbia hirta with 2C = 0.77 pg and Euphorbia hyssopifolia with 2C = 1.41 pg. After FISH procedures, Euphorbia hirta, and Euphorbia hyssopifolia presented three and four pairs of terminal 45S rDNA sites, respectively, colocalizing with CMA+ heterochromatic blocks, besides only one interstitial pair of 5S rDNA signals. Additionally, the maximum number of active NORs agreed with the total number of observed 45S rDNA sites. This work represents the first analysis using FISH in the subfamily Euphorbioideae, revealing a significant number of chromosomal markers, which may be very helpful to understand evolutionary patterns among Euphorbia species.

Quillworts from the Amazon: A multidisciplinary populational study on Isoetes serracarajensis and Isoetes cangae

Isoetes are ancient quillworts members of the only genus of the order Isoetales. The genus is slow evolving but is resilient, and widespread worldwide. Two recently described species occur in the Eastern Brazilian Amazon, Isoetes serracarajensis and Isoetes cangae. They are found in the ironstone grasslands known as Canga. While I. serracarajensis is present mostly in seasonal water bodies, I. cangae is known to occur in a single permanent lake at the South mountain range. In this work, we undertake an extensive morphological, physiological and genetic characterization of both species to establish species boundaries and better understand the morphological and genetic features of these two species. Our results indicate that the morphological differentiation of the species is subtle and requires a quantitative assessment of morphological elements of the megaspore for diagnosis. We did not detect differences in microspore output, but morphological peculiarities may establish a reproductive barrier. Additionally, genetic analysis using DNA barcodes and whole chloroplast genomes indicate that although the plants are genetically very similar both approaches provide diagnostic characters. There was no indication of population structuring I. serracarajensis. These results set the basis for a deeper understanding of the evolution of the Isoetes genus.