Carlos Augusto Colombo

Structure of genetic diversity among common bean ( Phaseolus vulgaris L.) varieties of Mesoamerican and Andean origins using new developed microsatellite markers

A common bean genomic library was constructed using the ‘IAC-UNA’ variety enriched for (CT) and (GT) for microsatellite motifs. From 1,209 sequenced clones, 714 showed microsatellites distributed over 471 simple and 243 compound motifs. GA/CT and GT/CA were the most frequent motifs found among these sequences. A total of 123 microsatellites has been characterized. Out of these, 87 were polymorphic (73.7%), 33 monomorphic (26.8%), and 3 (2.4%) did not amplify at all. In a sample of 20 common bean materials selected from the Agronomic Institute Germplasm Bank, the number of alleles per locus varied 2–9, with an average of 2.82. The polymorphic information content (PIC) of each marker varied from 0.05 to 0.83, with a 0.45 average value. Cluster and principal coordinate analysis of the microsatellite data were consistent with the original assignment of the germplasm accessions into the Andean and Mesoamerican gene pools of common bean. Low polymorphism levels detected could be associated with the domestication process. These microsatellites could be a valuable resource for the bean community because of their use as new markers for genetic studies.

An EST-based analysis identifies new genes and reveals distinctive gene expression features of Coffea arabica and Coffea canephora

BackgroundCoffee is one of the worlds most important crops; it is consumed worldwide and plays a significant role in the economy of producing countries. Coffea arabica and C. canephora are responsible for 70 and 30% of commercial production, respectively. C. arabica is an allotetraploid from a recent hybridization of the diploid species, C. canephora and C. eugenioides. C. arabica has lower genetic diversity and results in a higher quality beverage than C. canephora. Research initiatives have been launched to produce genomic and transcriptomic data about Coffea spp. as a strategy to improve breeding efficiency.ResultsAssembling the expressed sequence tags (ESTs) of C. arabica and C. canephora produced by the Brazilian Coffee Genome Project and the Nestlé-Cornell Consortium revealed 32,007 clusters of C. arabica and 16,665 clusters of C. canephora. We detected different GC3 profiles between these species that are related to their genome structure and mating system. BLAST analysis revealed similarities between coffee and grape (Vitis vinifera) genes. Using KA/KS analysis, we identified coffee genes under purifying and positive selection. Protein domain and gene ontology analyses suggested differences between Coffea spp. data, mainly in relation to complex sugar synthases and nucleotide binding proteins. OrthoMCL was used to identify specific and prevalent coffee protein families when compared to five other plant species. Among the interesting families annotated are new cystatins, glycine-rich proteins and RALF-like peptides. Hierarchical clustering was used to independently group C. arabica and C. canephora expression clusters according to expression data extracted from EST libraries, resulting in the identification of differentially expressed genes. Based on these results, we emphasize gene annotation and discuss plant defenses, abiotic stress and cup quality-related functional categories.ConclusionWe present the first comprehensive genome-wide transcript profile study of C. arabica and C. canephora, which can be freely assessed by the scientific community at http://www.lge.ibi.unicamp.br/coffea. Our data reveal the presence of species-specific/prevalent genes in coffee that may help to explain particular characteristics of these two crops. The identification of differentially expressed transcripts offers a starting point for the correlation between gene expression profiles and Coffea spp. developmental traits, providing valuable insights for coffee breeding and biotechnology, especially concerning sugar metabolism and stress tolerance.

A High-Throughput Data Mining of Single Nucleotide Polymorphisms in Coffea Species Expressed Sequence Tags Suggests Differential Homeologous Gene Expression in the Allotetraploid Coffea arabica

Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed.

Diversity within American cassava germ plasm based on RAPD markers

This work focuses on the genetic diversity of American cassava through RAPD molecular markers. The 126 genotypes studied were distributed on four geographical levels ranging from local to continental. Samples included ethnocultivars from the Santa Isabel community in the Brazilian Amazon, local cultivars collected in the State of Sao Paulo, native accessions from very diverse Brazilian regions, and representative accessions from the Centro Internacional de Agricultura Tropical (CIAT) core collection. Eighty-eight polymorphic bands were analyzed. Results revealed the weak genetic structure of the cassava analyzed. The pattern formed by the first two axes of the principal coordinates analysis (PCoA) revealed an overlapping of the Sao Paulo State genotype, the Brazilian group and the core collection accessions. The Santa Isabel ethnocultures formed a separate group. The weak genetic structure of cassava can be explained by the common practice of exchanging botanical material among small producers as well as by recombinations among genotypes. When the genotypes were analyzed using climatic data, the sample sites were found to be structured according to temperature and precipitation. RAPD markers proved very useful in the genetic diversity study, resulting in important implications for cassava germ plasm collections and genetic breeding.

Genetic diversity and structure of Ethiopian, Yemen and Brazilian Coffea arabica L. accessions using microsatellites markers

Genetic diversity among 115 coffee accessions from the Coffea Germplasm Collection of IAC was assessed using SSR markers. The germplasm represents 73 accessions of Coffea arabica derived from spontaneous and subspontaneous plants in Ethiopia and Eritrea, species center of origin and diversity, 13 commercial cultivars of C. arabica developed by the Breeding Program of IAC, 1 accession of C. arabica cv. ‘Geisha’, 13 accessions of C. arabica from Yemen, 5 accessions of C. eugenioides, 4 accessions of C. racemosa and 6 accessions of C. canephora. Genetic analysis was performed using average number of alleles per locus (A), proportion of polymorphic loci (P), Shannon’s genetic index (H′ and G′ST) and clustering analysis. All evaluated species were distinguished by a cluster analysis based on Jaccard’s coefficient. Differentiation between the cultivated plants of C. arabica and accessions derived from spontaneous and subspontaneous plants was observed. Spontaneous and subspontaneous accessions from Ethiopia were separated according to the geographical origin: east and west of the Great Rift Valley. Cultivated plants showed a low genetic diversity with a division in two groups: accessions from Yemen (H′=0,028) and Brazilian commercial cultivars (H′=0,030). The results agreed with previously reported narrow genetic basis of cultivated plants of C. arabica and supported the hypotheses about domestication of the species. This study also showed a significant genetic diversity among accessions from Ethiopia and Eritrea present in the Germplasm Collection of IAC. This diversity is specially observed in accessions from Sidamo (H′=0,143), Kaffa (H′=0,142) and Illubabor (H′=0,147) indicating their importance as source of genetic variability for coffee breeding programs.

Genetic diversity of cultivated Coffea arabica inbred lines assessed by RAPD, AFLP and SSR marker systems

One of the greatest problems in Coffea arabica breeding is identifying precisely any inbred line, based only on botanical and agronomical descriptors, because of the reduced genetic variability of the species, close pedigree origin, which results in small phenotypic variation. Recently, molecular markers have been used for plant germplasm characterization and identification in several commercial species. This work evaluates the reliability of three marker systems: RAPD, AFLP and SSR, to characterize the genetic variability of commercially-used Coffea inbred lines developed by the Instituto Agronomico (IAC), and their potential for cultivar identification. All methods identified polymorphisms among the cultivars. The genetic diversity recognized by the methods is very similar, although is very narrow. RAPD and SSR marker systems grouped more efficiently the evaluated cultivars according to parental origin. None of the methods allowed inbred line identification. Therefore for varietal protection, it would be necessary using a combination of botanical, agronomical and molecular markers descriptors for precise cultivar identification.

The genetic structure and mating system of Acrocomia aculeata (Arecaceae)

Acrocomia aculeata is a perennial, fruit-producing palm tree, native to tropical forests. Its fruits have spurred interest because of their significant potential for use in the cosmetic industry and as feedstock for biofuel. In the present study, the genetic structure and mating system in Acrocomia aculeata were analyzed, using eight nuclear micro-satellite loci and samples from São Paulo and Minas Gerais states, Brazil. By means of Bayesian analysis, these populations were clustered into two or three groups. A high multilocus outcrossing rate suggests that outcrosses were predominant, although a certain degree of biparental inbreeding also occurred. Thus, although monoecious and self-compatible, there is every indication that A. aculeata bears a mixed reproductive system, with a predominance of outcrossing. Given the genetic structure revealed hereby, future conservation strategies and germplasm collecting should be focussed on sampling and preserving individuals from different clusters.

Molecular characterization and genetic divergence of Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae) on different crops and growing areas

Knowledge on the genetic variation of populations of Bemisia tabaci (Genn.) can improve the understanding of genetic diversity found in their biotypes and, consequently, offer guidelines for its management. In this study, the molecular characterization was performed and genetic diversity data were obtained for this insect from three regions of Brazil on different crops [cotton and soybean (Mato Grosso - MT); cabbage (Distrito Federal - DF); soybean and potato (São Paulo - SP)], using RAPD markers. RAPD analysis indicated 80.6% polymorphic loci and the average genetic similarity obtained by the Jaccard coefficient was 0.67. The whitefly populations collected on potato (SP) and soybean (MT) had higher genetic diversity values (0.75 and 0.72, respectively). Shannons index (Ho) showed higher values for potato and soybean (SP e MT), and a smaller value for cabbage (DF). A high genetic divergence within and among the collected populations occurred, structured according to the regions of collection. Moreover, the great genetic similarity observed between potato (SP) and soybean (SP) populations suggested that both belong to the same biotype B and reinforces the polyphagous behavior of the species.

Transposable elements in Coffea (Gentianales: Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and Populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK β-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted.

PERFORMANCE OF GINGER GRASS (Lippia alba) FOR TRAITS RELATED TO THE PRODUCTION OF ESSENTIAL OIL

Lippia alba (Verbenaceae) is a shrub whose essential oil has important biological, pharmacological, and aromatizing properties. To reach the sustained cultivation of new species with economic potential, the present study aimed to evaluate L. alba performance for fresh leaf matter (FM), leaf dry matter (DM), virus symptoms (VS - Cucumber mosaic virus, CMV), oil yield (OY), and oil chemical composition (OC), and to evaluate DM stability and adaptability. Ten genotypes of four chemical groups (chemotypes) were evaluated in six experiments designed as randomized blocks with two plants per plot, over the whole State of Sao Paulo, Brazil. A 2.0 x 0.4 x 1.6 m spacing was used in Monte Alegre do Sul and Pindorama, while a 1.0 x 0.4 x 0.6 m spacing was used in Campinas, where four experiments were established under different irrigation and fertilization conditions. The genotype effect was significant (p < 0.05) for all traits evaluated, with high leaf productivity of IAC-16 (citral chemotype), best OY means in the linalool and limonene/carvone chemotypes, and susceptibility of the latter chemotype to CMV. The genotype performance oscillations in the six environments were significant for FM and DM, and despite their significance for phytochemical traits (OY and OC), they were of low magnitude. No qualitative variation was detected for OC. The IAC-2 (linalool) and IAC-13 (limonene/carvone) genotypes showed high stability and wide adaptability, and are recommended to establish initial cultivations of this species. This research also indicated genetic sources to start Lippia alba genetic breeding programs.