Expression of stress-related genes in zebrawood (Astronium fraxinifolium, Anacardiaceae) seedlings following germination in microgravity.

Seeds of a tropical tree species from Brazil, Astronium fraxinifolium, or zebrawood, were germinated, for the first time in microgravity, aboard the International Space Station for nine days. Following three days of subsequent growth under normal terrestrial gravitational conditions, greater root length and numbers of secondary roots was observed in the microgravity-treated seedlings compared to terrestrially germinated controls. Suppression subtractive hybridization of cDNA and EST analysis were used to detect differential gene expression in the microgravity-treated seedlings in comparison to those initially grown in normal gravity (forward subtraction). Despite their return to, and growth in normal gravity, the subtracted library derived from microgravity-treated seedlings was enriched in known microgravity stress-related ESTs, corresponding to large and small heat shock proteins, 14-3-3-like protein, polyubiquitin, and proteins involved in glutathione metabolism. In contrast, the reverse-subtracted library contained a comparatively greater variety of general metabolism-related ESTs, but was also enriched for peroxidase, possibly indicating the suppression of this protein in the microgravity-treated seedlings. Following continued growth for 30 days, higher concentrations of total chlorophyll were detected in the microgravity-exposed seedlings.

Shotgun sequencing for microsatellite identification in Ilex paraguariensis (Aquifoliaceae).

PREMISE OF THE STUDY: Ilex paraguariensis is a native tree species from Brazil, Argentina, and Paraguay that is used in the production of beverages, medicines, and cosmetics. Primers flanking microsatellites were developed to investigate genetic parameters in the species. • METHODS AND RESULTS: Using microsatellites cloned from an I. paraguariensis shotgun genomic library, 25 pairs of primers were designed and synthesized. Levels of polymorphism were evaluated in 24 individuals from two populations. Twenty loci were polymorphic, and an average of 4.8 and 4.5 alleles per locus were detected in the two populations, respectively. The mean observed heterozygosity was lower than the expected heterozygosity (0.54 vs. 0.60), indicating a departure from Hardy-Weinberg equilibrium and suggesting endogamy in both populations. • CONCLUSIONS: The reported set of markers is highly informative and constitutes a powerful tool for the development of genetic characterization studies in I. paraguariensis.

Development of expressed sequence tag and expressed sequence tag-simple sequence repeat marker resources for Musa acuminata.

BACKGROUND AND AIMS: Banana (Musa acuminata) is a crop contributing to global food security. Many varieties lack resistance to biotic stresses, due to sterility and narrow genetic background. The objective of this study was to develop an expressed sequence tag (EST) database of transcripts expressed during compatible and incompatible banana-Mycosphaerella fijiensis (Mf) interactions. Black leaf streak disease (BLSD), caused by Mf, is a destructive disease of banana. Microsatellite markers were developed as a resource for crop improvement. METHODOLOGY: cDNA libraries were constructed from in vitro-infected leaves from BLSD-resistant M. acuminata ssp. burmaniccoides Calcutta 4 (MAC4) and susceptible M. acuminata cv. Cavendish Grande Naine (MACV). Clones were 5'-end Sanger sequenced, ESTs assembled with TGICL and unigenes annotated using BLAST, Blast2GO and InterProScan. Mreps was used to screen for simple sequence repeats (SSRs), with markers evaluated for polymorphism using 20 diploid (AA) M. acuminata accessions contrasting in resistance to Mycosphaerella leaf spot diseases. PRINCIPAL RESULTS: A total of 9333 high-quality ESTs were obtained for MAC4 and 3964 for MACV, which assembled into 3995 unigenes. Of these, 2592 displayed homology to genes encoding proteins with known or putative function, and 266 to genes encoding proteins with unknown function. Gene ontology (GO) classification identified 543 GO terms, 2300 unigenes were assigned to EuKaryotic orthologous group categories and 312 mapped to Kyoto Encyclopedia of Genes and Genomes pathways. A total of 624 SSR loci were identified, with trinucleotide repeat motifs the most abundant in MAC4 (54.1 %) and MACV (57.6 %). Polymorphism across M. acuminata accessions was observed with 75 markers. Alleles per polymorphic locus ranged from 2 to 8, totalling 289. The polymorphism information content ranged from 0.08 to 0.81. CONCLUSIONS: This EST collection offers a resource for studying functional genes, including transcripts expressed in banana-Mf interactions. Markers are applicable for genetic mapping, diversity characterization and marker-assisted breeding.

Isolation and characterization of microsatellite loci in Cabralea canjerana (Meliaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed in the native Neotropical tree species Cabralea canjerana (Vell.) Mart. (Meliaceae) to study population genetics in some Atlantic Forest fragments of Brazil. This species is suitable for use in reforestation and is commercially important due to its high-quality wood. METHODS AND RESULTS: In this study, eight microsatellite loci were isolated from an enriched C. canjerana genomic library. These are the first microsatellite loci described for this genus. Genetic diversity analyses were carried out using 30 individuals and six polymorphic loci. An average of 11.2 alleles was found, and the observed heterozygosity was 0.65. CONCLUSIONS: The microsatellite markers described here are valuable tools for further population genetic studies of the species.

Characterization of novel microsatellite markers in Musa acuminata subsp. burmannicoides, var. Calcutta 4.

BACKGROUND: Banana is a nutritionally important crop across tropical and sub-tropical countries in sub-Saharan Africa, Central and South America and Asia. Although cultivars have evolved from diploid, triploid and tetraploid wild Asian species of Musa acuminata (A genome) and Musa balbisiana (B genome), many of today's commercial cultivars are sterile triploids or diploids, with fruit developing via parthenocarpy. As a result of restricted genetic variation, improvement has been limited, resulting in a crop frequently lacking resistance to pests and disease. Considering the importance of molecular tools to facilitate development of disease resistant genotypes, the objectives of this study were to develop polymorphic microsatellite markers from BAC clone sequences for M. acuminata subsp. burmannicoides, var. Calcutta 4. This wild diploid species is used as a donor cultivar in breeding programs as a source of resistance to diverse biotic stresses. FINDINGS: Microsatellite sequences were identified from five Calcutta 4 BAC consensi datasets. Specific primers were designed for 41 loci. Isolated di-nucleotide repeat motifs were the most abundant, followed by tri-nucleotides. From 33 tested loci, 20 displayed polymorphism when screened across 21 diploid M. acuminata accessions, contrasting in resistance to Sigatoka diseases. The number of alleles per SSR locus ranged from two to four, with a total of 56. Six repeat classes were identified, with di-nucleotides the most abundant. Expected heterozygosity values for polymorphic markers ranged from 0.31 to 0.75. CONCLUSIONS: This is the first report identifying polymorphic microsatellite markers from M. acuminata subsp. burmannicoides, var. Calcutta 4 across accessions contrasting in resistance to Sigatoka diseases. These BAC-derived polymorphic microsatellite markers are a useful resource for banana, applicable for genetic map development, germplasm characterization, evolutionary studies and marker assisted selection for traits.

Insights into the Musa genome: syntenic relationships to rice and between Musa species.

BACKGROUND: Musa species (Zingiberaceae, Zingiberales) including bananas and plantains are collectively the fourth most important crop in developing countries. Knowledge concerning Musa genome structure and the origin of distinct cultivars has greatly increased over the last few years. Until now, however, no large-scale analyses of Musa genomic sequence have been conducted. This study compares genomic sequence in two Musa species with orthologous regions in the rice genome. RESULTS: We produced 1.4 Mb of Musa sequence from 13 BAC clones, annotated and analyzed them along with 4 previously sequenced BACs. The 443 predicted genes revealed that Zingiberales genes share GC content and distribution characteristics with eudicot and Poaceae genomes. Comparison with rice revealed microsynteny regions that have persisted since the divergence of the Commelinid orders Poales and Zingiberales at least 117 Mya. The previously hypothesized large-scale duplication event in the common ancestor of major cereal lineages within the Poaceae was verified. The divergence time distributions for Musa-Zingiber (Zingiberaceae, Zingiberales) orthologs and paralogs provide strong evidence for a large-scale duplication event in the Musa lineage after its divergence from the Zingiberaceae approximately 61 Mya. Comparisons of genomic regions from M. acuminata and M. balbisiana revealed highly conserved genome structure, and indicated that these genomes diverged circa 4.6 Mya. CONCLUSION: These results point to the utility of comparative analyses between distantly-related monocot species such as rice and Musa for improving our understanding of monocot genome evolution. Sequencing the genome of M. acuminata would provide a strong foundation for comparative genomics in the monocots. In addition a genome sequence would aid genomic and genetic analyses of cultivated Musa polyploid genotypes in research aimed at localizing and cloning genes controlling important agronomic traits for breeding purposes.

Genetic structure and mating system of Manilkara huberi (Ducke) A. Chev., a heavily logged Amazonian timber species.

In this work, we report on the population genetic structure of the endangered tree species Manilkara huberi, an Amazonian tree species intensely exploited due to the high density and resistance of its wood. We investigated the patterns of spatial distribution, genetic structure, and mating system using 7 microsatellite loci and here discuss the consequences for conservation and management of the species. To examine the population genetic structure, 481 adult trees and 810 seedlings were sampled from an area of 200 ha from a natural population in FLONA Tapajós, PA, Brazil. We found relatively high and consistent inbreeding levels (intrapopulation fixation index [f] 0.175 and 0.240) and a significant spatial genetic structure up to a radius of approximately 300 m, most likely due to a limited seed and pollen flow. The multilocus (tm) population outcrossing rate was high (0.995), suggesting that the species is predominantly allogamous with a pollen flow restricted to 47 m. These results suggest that M. huberi is spatially structured, consistent with a model of isolation by distance. Fragmentation may therefore cause the loss of subpopulations, suggesting that management programs for production and conservation should include large areas. The genetic data also revealed that for ex situ conservation, seeds should be collected from more than 175 maternal trees, in order to keep an effective population size of 500. Furthermore, as the species is widely distributed across the Amazon Forest, samples should include several populations in order to represent the highest genetic diversity possible. These results provide a blueprint to guide the production and conservation management policies of this valuable timber species.