Eduardo de Andrade Bressan

Genetic diversity in Brazilian sweet potato (Ipomoea batatas (L.) Lam., Solanales, Convolvulaceae) landraces assessed with microsatellite markers

We used simple sequence repeat (SSR) markers to investigate the genetic diversity of 78 sweet potato (Ipomoea batatas) accessions (58 landraces and 20 putative clones) from traditional agricultural households from 19 local communities in the Vale do Ribeira, Sao Paulo, Brazil. Eight SSR loci were assessed using 6% (w/v) polyacrylamide gels stained with silver nitrate and the accessions genotyped considering the presence or absence of bands. The results were subjected to analysis of molecular variance (AMOVA), and cluster and principal coordinate analyses. Spatial structure was assessed using Mantels test to compare genetic and geographic distances. Each primer pair generated between three and ten clearly scorable polymorphic fragments. Cluster analyses showed a Jaccards index from 0.3 to 1.0, indicating high genetic and intravarietal diversity. Accessions from all 19 communities were not spatially structured (r = 0.15, p < 0.054), with AMOVA indicating that most of the variability (58.2%) was distributed within households and only 18.1% of the variability was distributed between households within communities. The outcrossing mating system of sweet potato, and anthropic factors such as selection of different varieties and their maintenance within household small plots and home gardens, as well as an extensive exchange system between agriculturists, may all be contributing to these results.

Genetic characterization of cassava ( Manihot esculenta ) landraces in Brazil assessed with simple sequence repeats

Based on nine microsatellite loci, the aim of this study was to appraise the genetic diversity of 42 cassava (Manihot esculenta) landraces from selected regions in Brazil, and examine how this variety is distributed according to origin in several municipalities in the states of Minas Gerais, São Paulo, Mato Grosso do Sul, Amazonas and Mato Grosso. High diversity values were found among the five above-mentioned regions, with 3.3 alleles per locus on an average, a high percentage of polymorphic loci varying from 88.8% to 100%, an average of 0.265 for observed heterozygosity and 0.570 for gene diversity. Most genetic diversity was concentrated within the regions themselves (HS = 0.52). Cluster analysis and principal component based scatter plotting showed greater similarity among landraces from São Paulo, Mato Grosso do Sul and Amazonas, whereas those from Minas Gerais were clustered into a sub-group within this group. The plants from Mato Grosso, mostly collected in the municipality of General Carneiro, provided the highest differentiation. The migration of human populations is one among the possible reasons for this closer resemblance or greater disparity among plants from the various regions.

Phenology and morphological diversity of sweet potato (Ipomoea batatas) landraces of the Vale do Ribeira

The phenotypic diversity of sweet potato (Ipomoea batatas) landraces was assessed using morphological traits, verifying how this diversity is distributed among the households and settlements of the Vale do Ribeira, Brazil. A total of 74 accessions, involving 53 landraces, collected from 30 households distributed among 18 settlements that practice traditional agriculture in the municipalities of Iguape, Ilha Comprida, and Cananeia, as well as four commercial varieties acquired in markets of Iguape and Piracicaba, were evaluated under an ex situ experimental condition in Piracicaba, SP, Brazil. Nine phenological and floral descriptors, nine morphological vegetative aerial descriptors and five storage root traits were recorded. The 14 aerial vegetative and root descriptors were evaluated as binary data, totaling 74 attributes. Cluster analyses were made using the Jaccard similarity index and the UPGMA (unweighted pair group method with arithmetic mean) agglomerative method. Binary data was also submitted to a variance analysis (AMOVA). No defined groups were observed, indicating that the diversity of the landraces is not structured in space, but considerable morphological variation was found in this area (Jaccard similarity index varying from 0.12 to 1.0). Most of the variability occurred within households (64.4%), followed by the distribution among households within settlements (27.1%) and among settlements (8.4%). Thus, the traditional agriculturists of Vale do Ribeira maintain a high morphological diversity for sweet potato within their households, which can be assumed to be produced by the outcrossing mating system of this species and somatic mutation events, as well as the exchange system at local and regional levels.

Development of microsatellite primers for Jatropha curcas (Euphorbiaceae) and transferability to congeners

PREMISE OF THE STUDY Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. METHODS AND RESULTS Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. CONCLUSIONS The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.

CTAB methods for DNA extraction of sweetpotato for microsatellite analysis

Microsatellite markers have proved to be useful in genetic diversity assessments of sweetpotato (Ipomoea batatas) but practical DNA extraction methods to ensure good quality and quantity DNA for these studies are yet to be established. This study compares the efficiency of three modified methodologies for DNA extraction of six sweetpotato landraces using the CTAB extraction buffer in regard to quantity and purity of DNA quantification and microsatellite band patterns. All methodologies yielded satisfactory results, but the method based in leaf tissue macerated in liquid nitrogen was deemed more adequate because of its simplicity and lower cost. However, the method based in dry leaf tissue was considered more advantageous, first because elicits practicability in the plant acquisition and drying process, especially when the collection is performed in situ, and also because its simplicity makes possible the cold storage of the dry, ground samples for future DNA extractions.

Morphological variation and isozyme diversity in Dioscorea alata L. landraces from Vale do Ribeira, Brazil

Traditional growers of the Vale do Ribeira, Sao Paulo State, grow and make use of several D. alata landraces. This study assessed the genetic diversity of 16 landraces, using isozymatic and morphological markers and comparing them with 19 commercial varieties of D. alata. Their distribution in different levels of organization such as households and communities of the Vale do Ribeira was evaluated. Isozymatic analyses were performed with polyacrylamide (six systems) and starch gels (one system), while the morphological analyses were carried out with 24 traits. Due to the polyploid nature of this species, the isozymatic bands were scored as binary data. Morphological traits were also scored as binary data. Principal coordinates and cluster analyses were conducted for both markers, using for the later the Jaccard´s similarity coefficient and UPGMA method. The separation of the landraces from the commercial varieties, which showed lower genetic diversity, was reported for both markers. No correlation between genetic and geographical distances was found for both data, which suggests that the observed variability is not spatially structured. Also, no correlation was found between both markers. The analysis of molecular variance (AMOVA) indicated that genetic diversity was mainly found within households for both isozymatic (54%) and morphological (70%) markers. The results obtained for both markers revealed the importance of traditional agriculturists in the Vale do Ribeira in maintaining high diversity for D. alata, even higher than the varieties commercialized in Sao Paulo State, contributing for the in situ/on farm conservation of this crop.

Assessing the genetic structure of Oryza glumaepatula populations with isozyme markers

To assess the genetic diversity and genetic structure parame ters, nine populations of Oryza glumaepatula from the Amazon biome, four from the Pantanal biome, and one collected at Rio Xingu, Mato Grosso, totaling 14 populations and 333 individuals were studied with isozyme markers. Six loci were evaluated showing a moderate allozyme variability ( A = 1.21, P = 20.7%, H o = 0.005, H e = 0.060). The populations from the Pantanal biome showed higher diversity levels than the Amazon biome. High genetic differentiation among the populations, expected for self-fertilizing species, was observed (F ST=0.763), with lower differentiation found among the Pantanal populations

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

BackgroundProper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species.ResultsTotal DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with Eco RI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA.ConclusionsTissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.

Genetic diversity of American wild rice species

Studies on genetic diversity and genetic structure of natural populations are important in order to define strategies for in situ and ex situ conservation actions and for plant pre-breeding programs. Aiming to assess the genetic diversity and genetic structure of three wild American Oryza species with isozyme markers, 14 populations of the diploid O. glumaepatula (AglAgl), 11 populations of the tetraploid O. grandiglumis (CCDD) and five populations of the also tetraploid O. latifolia (CCDD) were studied. They were all originated from Rio Paraguay hydrographic basin and the Amazon. Four enzymes were used and they gave 40 polymorphic bands. The most polymorphic species was O. glumaepatula, followed by O. latifolia and O. grandiglumis. A cluster analysis with the Jaccard similarity coefficient separated the diploid from the two tetraploid species, and also the two tetraploid species. This separation was also evident on a scatter plot from a principal component analysis, suggesting that they should be treated as two separate species, although further studies are necessary to provide support for this affirmative. The AMOVA analyses showed a high intrapopulational variability for O. latifolia (67.6%) and O. grandiglumis (52.2%), when compared to their interpopulational variability (32.4% and 47.8%, respectively), which suggests the hypothesis of a higher degree of outcrossing events within these species. When studying the correlation between the Jaccard dissimilarity coefficient and geographic distances, a spatial genetic structure was observed for O. glumaepatula only. These results are important for defining strategies of both in situ and ex situ conservation.