F. W. Cidade

Genetic variation in polyploid forage grass: Assessing the molecular genetic variability in the Paspalum genus

BackgroundPaspalum (Poaceae) is an important genus of the tribe Paniceae, which includes several species of economic importance for foraging, turf and ornamental purposes, and has a complex taxonomical classification. Because of the widespread interest in several species of this genus, many accessions have been conserved in germplasm banks and distributed throughout various countries around the world, mainly for the purposes of cultivar development and cytogenetic studies. Correct identification of germplasms and quantification of their variability are necessary for the proper development of conservation and breeding programs. Evaluation of microsatellite markers in different species of Paspalum conserved in a germplasm bank allowed assessment of the genetic differences among them and assisted in their proper botanical classification.ResultsSeventeen new polymorphic microsatellites were developed for Paspalum atratum Swallen and Paspalum notatum Flüggé, twelve of which were transferred to 35 Paspalum species and used to evaluate their variability. Variable degrees of polymorphism were observed within the species. Based on distance-based methods and a Bayesian clustering approach, the accessions were divided into three main species groups, two of which corresponded to the previously described Plicatula and Notata Paspalum groups. In more accurate analyses of P. notatum accessions, the genetic variation that was evaluated used thirty simple sequence repeat (SSR) loci and revealed seven distinct genetic groups and a correspondence of these groups to the three botanical varieties of the species (P. notatum var. notatum, P. notatum var. saurae and P. notatum var. latiflorum).ConclusionsThe molecular genetic approach employed in this study was able to distinguish many of the different taxa examined, except for species that belong to the Plicatula group, which has historically been recognized as a highly complex group. Our molecular genetic approach represents a valuable tool for species identification in the initial assessment of germplasm as well as for characterization, conservation and successful species hybridization.

Genetic structure and phenotypic variation in wild populations of the medicinal tetraploid species Bromelia antiacantha (Bromeliaceae)

UNLABELLED PREMISE OF THE STUDY The patterns of genetic structure in plant populations are mainly related to the species life history and breeding system, and knowledge of these patterns is necessary for the management, use, and conservation of biological diversity. Polyploidy is considered an important mode of evolution in plants, but few studies have evaluated genetic structure of polyploid populations. We studied the patterns of genetic structure and morphological variation of Bromelia antiacantha (Bromeliaceae) populations, a polyploid terrestrial species. • METHODS Microsatellite markers and morphological analyses were used to explore patterns of genetic and morphological diversity in wild populations of B. antiacantha. • KEY RESULTS The results of our simple-sequence repeat analyses supported that B. antiacantha is a polyploid species. The inbreeding coefficients were high and significant in all populations (F(IS) = 0.431), indicating homozygote excess. Bromelia antiacantha showed high levels of genetic differentiation among populations (F(ST) = 0.224) and therefore was highly structured. High morphological variation was observed in fruit phenotypic traits in the populations studied. • CONCLUSIONS The levels of genetic diversity and the pattern of the populations structure may be related to the low recruitment of seeds, clonal reproduction, and the populations colonization history. The genetic and morphological variability displayed in this study are important issues in planning the conservation and exploitation of this resource in a sustainable way.

Isolation and characterization of microsatellite loci in Pitcairnia albiflos (Bromeliaceae), an endemic bromeliad from the Atlantic Rainforest, and cross-amplification in other species

Eight microsatellite markers were isolated from Pitcairnia albiflos, an endangered endemic bromeliad species restricted to ‘inselberg’ rock outcrops in the state of Rio de Janeiro, Brazil. The number of alleles observed for each locus ranged from two to 12. Average observed and expected heterozygosities were 0.408 and 0.663, respectively. The cross‐amplification test in 16 taxa suggests that the markers will be useful in numerous related bromeliad species. The loci will be used to study genetic structure and reproductive biology in fragmented inselberg populations and the origin and maintenance of barriers to gene flow between sympatric Pitcairnia species.

Isolation and characterization of microsatellite loci in the black pepper, Piper nigrum L. (piperaceae)

The black pepper, Piper nigrum L., which originated in Índia, is the World’s most important commercial spice. Brazil has a germplasm collection of this species preserved at the Brazilian Agricultural Research Corporation (Embrapa—Eastern Amazonia) where efforts are being made to generation information on the patterns of genetic variation and develop strategies for conservation and management of black pepper. Molecular markers of the SSR type are powerful tools for the description of material preserved in genetic resources banks, due to characteristics such as high levels of polymorphism, codominance and Mendelian segregation. Given this, we developed nine microsatellite markers from an enriched library of Piper nigrum L. Twenty varieties clonal from the Brazilian germplasm collection were analyzed, and observed and expected heterozygosity values ranged over 0.11–1.00 and 0.47–0.87, respectively. The nine microsatellite loci characterized here will contribute to studies of genetic diversity and conservation of Piper nigrum L.

Isolation and characterization of microsatellite loci in Paspalum notatum Flüggé (Poaceae)

Paspalum notatum is a forage grass recognized as one of the major constituents of the native grasslands in the New World. The knowledge of the genetic diversity and structure of P. notatum populations is fundamental for the conservation and germplasm management of this species. About 11 microsatellite markers were isolated from P. notatum and characterized in 25 accessions. The average number of alleles per locus was 7.9 and the PIC ranged from 0.36 to 0.89. The data demonstrated that the most of markers are suitable to detect polymorphism and to study the genetic diversity in the P. notatum species. Moreover, the transferability of these microsatellite were tested on other three congeneric species.

Isolation and characterization of tri and tetranucleotide microsatellite markers for the tambaqui (Colossoma macropomum, Serrasalmidae, Characiformes)

We isolated 13 tri and tetranucleotide microsatellite markers for the species Colossoma macropomum that can be used in management programmes for this species of Amazon fish. This panel of microsatellite markers was used in the genotyping of 20 individual specimens collected in the lakes of Óbidos city, in the Brazilian Amazon. The number of alleles per locus varied from four to ten. The observed heterozygosity varied from 0.31 to 0.95. We observed no significant deviation from the expected Hardy–Weinberg equilibrium assumption. In the sample investigated, it was not possible to identify any significant linkage disequilibrium among the 78 possible loci pairs. In our analysis, we found no indication of genotyping error attributed to stutter bands, large allele dropout or null alleles.

MICROSATELLITE LOCI FOR PASPALUM ATRATUM (POACEAE) AND CROSS-AMPLIFICATION IN OTHER SPECIES

UNLABELLED PREMISE OF THE STUDY Paspalum atratum is a perennial, cespitose, tropical grass native to Central and South America. This species belongs to a polyploid complex (Plicatula group) little known at the genetic level. The characterized microsatellite markers provide new informative tools for further studies of the hybridization, mating systems, and structure of the population. • METHODS AND RESULTS Using the microsatellite-enriched library method, we isolated and characterized 19 microsatellite markers from P. atratum. Eleven of them were polymorphic, showing a variable degree of variation, while eight were monomorphic in the samples analyzed. Additionally, the transferability of these microsatellite markers was tested in other species. • CONCLUSIONS These results suggest that the characterized markers have enough discriminatory potential to be used in genetic characterizations of Paspalum taxa, which are based on an understanding of their mating systems and genetic structure, as well as in understanding the evolutionary processes involved in the evolution of groups of Paspalum.

Genetic diversity of Metrodorea nigra (Rutaceae) from a small forest remnant in Brazil assessed with microsatellite markers

Metrodorea nigra (Rutaceae) is an endemic Brazilian tree of great ecological importance, frequently found in the submontane regions of ombrophilous dense and semideciduous forests. This tree is useful for reforesting degraded areas and the wood can be employed in construction. We developed 12 microsatellite markers from a genomic library enriched for GA/CA repeats, for this species. Polymorphisms were assessed in 40 trees of a highly fragmented population found in Cravinhos, State of São Paulo, in southeastern Brazil. Among the 12 loci, 8 were polymorphic and only one had fixed alleles in this population. The number of alleles per locus and expected heterozygosity ranged from 2 to 11 and from 0.190 to 0.889, respectively. These results revealed moderate levels of genetic variation in M. nigra population when compared to other tropical species. Additionally, transferability of the 12 primers was tested in seven other Brazilian Rutaceae tree species (endemics: M. stipularis, Galipea jasminiflora, Esenbeckia leiocarpa and non-endemics: E. febrifuga, E. grandiflora, Balfourodendron riedelianum, Zanthoxylum riedelianum). Transferability ranged among species, but at least 8 loci (~67%) amplified in M. stipularis, demonstrating a high potential for transferring microsatellite markers between species of the same genus in the Rutaceae family.

New 24 polymorphic DNA microsatellite loci for the major malaria vector Anopheles darlingi and transpecies amplification with another anophelines

Anopheles darlingi is a major human malaria vector in the Neotropics. Twenty-four polymorphic microsatellite loci were isolated and characterized in 21–32 individuals collected in Coari (Amazonas, Brazil). The number of alleles per locus ranged from 4 to 11 (average of 7.667). The observed heterozygosity (HO) varied between 0.037 and 0.833 (average of 0.500), while the expected heterozygosity (HE) ranged from 0.177 to 0.871 (average of 0.723). Thirteen loci showed a significant deviation from HWE. No linkage disequilibrium was found between the loci.

Isolation and characterization of microsatellite loci in Sisyrinchium (Iridaceae) and cross amplification in other genera

Recent phylogenetic studies on Sisyrinchium strongly suggest that species classified in section Hydastylus and section Viperella belong to a single group of plants in recent adaptive radiation (Clade IV). These species neither present clear morphological differentiation among them nor show clear identification using DNA barcode markers. Thus, the main goal of this study was to develop a set of polymorphic microsatellite markers compatible for representative species of both sections to ensure variability that could be revealed by SSR markers. Therefore, microsatellite primers were isolated and characterized for Sisyrinchium palmifolium and S. marchioides. In addition, transferability of the developed primers was tested in Iridoideae, primarily in closely related species of Sisyrinchium. Sixteen microsatellite loci were developed from enriched genomic libraries, of which ten were polymorphic. GST values indicated higher differentiation among subpopulations of S. palmifolium than those from S. marchioides. Major transferability was obtained using primers isolated from S. marchioides. All primers exhibited higher rates of cross-amplification for species belonging to Clade IV of Sisyrinchium, as well as to the genera Calydorea and Herbertia. These developed microsatellite markers can be used as an efficient tool for characterization of genetic variability in species belonging to Iridoideae, as well as for studies on population dynamics, genetic structure, and mating system in other Sisyrinchium species.