Trends in biological data integration for the selection of enzymes and transcription factors related to cellulose and hemicellulose degradation in fungi.

Fungi are key players in biotechnological applications. Although several studies focusing on fungal diversity and genetics have been performed, many details of fungal biology remain unknown, including how cellulolytic enzymes are modulated within these organisms to allow changes in main plant cell wall compounds, cellulose and hemicellulose, and subsequent biomass conversion. With the advent and consolidation of DNA/RNA sequencing technology, different types of information can be generated at the genomic, structural and functional levels, including the gene expression profiles and regulatory mechanisms of these organisms, during degradation-induced conditions. This increase in data generation made rapid computational development necessary to deal with the large amounts of data generated. In this context, the origination of bioinformatics, a hybrid science integrating biological data with various techniques for information storage, distribution and analysis, was a fundamental step toward the current state-of-the-art in the postgenomic era. The possibility of integrating biological big data has facilitated exciting discoveries, including identifying novel mechanisms and more efficient enzymes, increasing yields, reducing costs and expanding opportunities in the bioprocess field. In this review, we summarize the current status and trends of the integration of different types of biological data through bioinformatics approaches for biological data analysis and enzyme selection.

"Integrative genomic analysis of the bioprospection of regulators and accessory enzymes associated with cellulose degradation in a filamentous fungus (Trichoderma harzianum)".

BACKGROUND: Unveiling fungal genome structure and function reveals the potential biotechnological use of fungi. Trichoderma harzianum is a powerful CAZyme-producing fungus. We studied the genomic regions in T. harzianum IOC3844 containing CAZyme genes, transcription factors and transporters. RESULTS: We used bioinformatics tools to mine the T. harzianum genome for potential genomics, transcriptomics, and exoproteomics data and coexpression networks. The DNA was sequenced by PacBio SMRT technology for multiomics data analysis and integration. In total, 1676 genes were annotated in the genomic regions analyzed; 222 were identified as CAZymes in T. harzianum IOC3844. When comparing transcriptome data under cellulose or glucose conditions, 114 genes were differentially expressed in cellulose, with 51 being CAZymes. CLR2, a transcription factor physically and phylogenetically conserved in Trichoderma spp., was differentially expressed under cellulose conditions. The genes induced/repressed under cellulose conditions included those important for plant biomass degradation, including CIP2 of the CE15 family and a copper-dependent LPMO of the AA9 family. CONCLUSIONS: Our results provide new insights into the relationship between genomic organization and hydrolytic enzyme expression and regulation in T. harzianum IOC3844. Our results can improve plant biomass degradation, which is fundamental for developing more efficient strains and/or enzymatic cocktails to produce hydrolytic enzymes.

High-Resolution Linkage Map With Allele Dosage Allows the Identification of Regions Governing Complex Traits and Apospory in Guinea Grass (Megathyrsus maximus).

Forage grasses are mainly used in animal feed to fatten cattle and dairy herds, and guinea grass (Megathyrsus maximus) is considered one of the most productive of the tropical forage crops that reproduce by seeds. Due to the recent process of domestication, this species has several genomic complexities, such as autotetraploidy and aposporous apomixis. Consequently, approaches that relate phenotypic and genotypic data are incipient. In this context, we built a linkage map with allele dosage and generated novel information of the genetic architecture of traits that are important for the breeding of M. maximus. From a full-sib progeny, a linkage map containing 858 single nucleotide polymorphism (SNP) markers with allele dosage information expected for an autotetraploid was obtained. The high genetic variability of the progeny allowed us to map 10 quantitative trait loci (QTLs) related to agronomic traits, such as regrowth capacity and total dry matter, and 36 QTLs related to nutritional quality, which were distributed among all homology groups (HGs). Various overlapping regions associated with the quantitative traits suggested QTL hotspots. In addition, we were able to map one locus that controls apospory (apo-locus) in HG II. A total of 55 different gene families involved in cellular metabolism and plant growth were identified from markers adjacent to the QTLs and APOSPORY locus using the Panicum virgatum genome as a reference in comparisons with the genomes of Arabidopsis thaliana and Oryza sativa. Our results provide a better understanding of the genetic basis of reproduction by apomixis and traits important for breeding programs that considerably influence animal productivity as well as the quality of meat and milk.

Coexpression and Transcriptome analyses identify active Apomixis-related genes in Paspalum notatum leaves.

BACKGROUND: Paspalum notatum exhibits both sexual and apomictic cytotypes and, thus, is considered a good model for studies of apomixis because it facilitates comparative approaches. In this work, transcriptome sequencing was used to compare contrasting P. notatum cytotypes to identify differential expression patterns and candidate genes involved in the regulation of expression of this trait. RESULTS: We built a comprehensive transcriptome using leaf and inflorescence from apomictic tetraploids and sexual diploids/tetraploids and a coexpression network based on pairwise correlations between transcript expression profiles. We identified genes exclusively expressed in each cytotype and genes differentially expressed between pairs of cytotypes. Gene Ontology enrichment analyses were performed to better interpret the data. We de novo assembled 114,306 reference transcripts. In total, 536 candidate genes possibly associated with apomixis were detected through statistical analyses of the differential expression data, and several interacting genes potentially linked to the apomixis-controlling region, genes that have already been reported in the literature, and their neighbors were transcriptionally related in the coexpression network. CONCLUSIONS: Apomixis is a highly desirable trait in modern agriculture due to the maintenance of the characteristics of the mother plant in the progeny. The reference transcriptome, candidate genes and their coexpression network identified in this work represent rich resources for future grass breeding programs.

Extremophiles as a Model of a Natural Ecosystem: Transcriptional Coordination of Genes Reveals Distinct Selective Responses of Plants Under Climate Change Scenarios.

The goal of this research was to generate networks of co-expressed genes to explore the genomic responses of Rhizophora mangle L. populations to contrasting environments and to use gene network analysis to investigate their capacity for adaptation in the face of historical and future perturbations and climatic changes. RNA sequencing data were generated for R. mangle samples collected under field conditions from contrasting climate zones in the equatorial and subtropical regions of Brazil. A gene co-expression network was constructed using Pearson's correlation coefficient, showing correlations among 78,364 transcriptionally coordinated genes. Each region exhibited two distinct network profiles; genes correlated with the oxidative stress response showed higher relative expression levels in subtropical samples than in equatorial samples, whereas genes correlated with the hyperosmotic salinity response, heat response and UV response had higher expression levels in the equatorial samples than in the subtropical samples. In total, 992 clusters had enriched ontology terms, which suggests that R. mangle is under higher stress in the equatorial region than in the subtropical region. Increased heat may thus pose a substantial risk to species diversity at the center of its distribution range in the Americas. This study, which was performed using trees in natural field conditions, allowed us to associate the specific responses of genes previously described in controlled environments with their responses to the local habitat where the species occurs. The study reveals the effects of contrasting environments on gene expression in R. mangle, shedding light on the different abiotic variables that may contribute to the genetic divergence previously described for the species through the use of simple sequence repeats (SSRs). These effects may result from two fundamental processes in evolution, namely, phenotypic plasticity and natural selection.

High-Resolution Genetic Map and QTL Analysis of Growth-Related Traits of Hevea brasiliensis Cultivated Under Suboptimal Temperature and Humidity Conditions.

Rubber tree (Hevea brasiliensis) cultivation is the main source of natural rubber worldwide and has been extended to areas with suboptimal climates and lengthy drought periods; this transition affects growth and latex production. High-density genetic maps with reliable markers support precise mapping of quantitative trait loci (QTL), which can help reveal the complex genome of the species, provide tools to enhance molecular breeding, and shorten the breeding cycle. In this study, QTL mapping of the stem diameter, tree height, and number of whorls was performed for a full-sibling population derived from a GT1 and RRIM701 cross. A total of 225 simple sequence repeats (SSRs) and 186 single-nucleotide polymorphism (SNP) markers were used to construct a base map with 18 linkage groups and to anchor 671 SNPs from genotyping by sequencing (GBS) to produce a very dense linkage map with small intervals between loci. The final map was composed of 1,079 markers, spanned 3,779.7 cM with an average marker density of 3.5 cM, and showed collinearity between markers from previous studies. Significant variation in phenotypic characteristics was found over a 59-month evaluation period with a total of 38 QTLs being identified through a composite interval mapping method. Linkage group 4 showed the greatest number of QTLs (7), with phenotypic explained values varying from 7.67 to 14.07%. Additionally, we estimated segregation patterns, dominance, and additive effects for each QTL. A total of 53 significant effects for stem diameter were observed, and these effects were mostly related to additivity in the GT1 clone. Associating accurate genome assemblies and genetic maps represents a promising strategy for identifying the genetic basis of phenotypic traits in rubber trees. Then, further research can benefit from the QTLs identified herein, providing a better understanding of the key determinant genes associated with growth of Hevea brasiliensis under limiting water conditions.

Genetic structure of two Prosopis species in Chaco areas: A lack of allelic diversity diagnosis and insights into the allelic conservation of the affected species.

The Gran Chaco is the largest continuous region of the South American dry forest, spanning Argentina, Paraguay, Bolivia, and Brazil. Prosopis rubriflora and Prosopis ruscifolia are typical tree species of chaquenian area forests, which have been subjected to continuous fragmentation caused by cattle raising. This study evaluated P. rubriflora and P. ruscifolia in areas with varying levels of disturbance. We investigated the contemporary genetic diversities of both species in areas with distinct anthropogenic disturbances. Even with a lower heterozygote frequency, disturbed areas can provide important storage for alleles, allowing the maintenance of diversity. The genetic diversity of P. rubriflora was surprisingly similar to that of P. ruscifolia (He = 0.59 and He = 0.60, respectively) even with very different distribution ranges of both species. However, P. ruscifolia exhibited a higher intrapopulation fixation index than P. rubriflora. P. rubriflora showed evidence of bottlenecking in 64% of the sampled areas, while P. ruscifolia showed such evidence in 36% of the sampled areas. Additionally, P. rubriflora had two distinct populations due to its disjunctive geographic distribution, whereas P. ruscifolia had a single population that exhibited few signs of population structure in some areas, possibly due to the main pollinators presenting a short range of dispersion. Our results suggest that 42 Chaco areas should be conserved to retain the minimum of 500 individuals necessary to maintain genetic diversity for 100-1,000 generations. This study improves our understanding of these two Prosopis species and provides information for the conservation of their genetic diversities.

Linkage Disequilibrium and Population Structure in Wild and Cultivated Populations of Rubber Tree (Hevea brasiliensis).

Among rubber tree species, which belong to the Hevea genus of the Euphorbiaceae family, Hevea brasiliensis (Willd. ex Adr.de Juss.) Muell. Arg. is the main commercial source of natural rubber production worldwide. Knowledge of the population structure and linkage disequilibrium (LD) of this species is essential for the efficient organization and exploitation of genetic resources. Here, we obtained single-nucleotide polymorphisms (SNPs) using a genotyping-by-sequencing (GBS) approach and then employed the SNPs for the following objectives: (i) to identify the positions of SNPs on a genetic map of a segregating mapping population, (ii) to evaluate the population structure of a germplasm collection, and (iii) to detect patterns of LD decay among chromosomes for future genetic association studies in rubber tree. A total of 626 genotypes, including both germplasm accessions (368) and individuals from a genetic mapping population (254), were genotyped. A total of 77,660 and 21,283 SNPs were detected by GBS in the germplasm and mapping populations, respectively. The mapping population, which was previously mapped, was constructed with 1,062 markers, among which only 576 SNPs came from GBS, reducing the average interval between two adjacent markers to 4.4 cM. SNPs from GBS genotyping were used for the analysis of genetic structure and LD estimation in the germplasm accessions. Two groups, which largely corresponded to the cultivated and wild populations, were detected using STRUCTURE and via principal coordinate analysis. LD analysis, also using the mapped SNPs, revealed that non-random associations varied along chromosomes, with regions of high LD interspersed with regions of low LD. Considering the length of the genetic map (4,693 cM) and the mean LD (0.49 for cultivated and 0.02 for wild populations), a large number of evenly spaced SNPs would be needed to perform genome-wide association studies in rubber tree, and the wilder the genotypes used, the more difficult the mapping saturation.

Population genetic structure, introgression, and hybridization in the genus Rhizophora along the Brazilian coast.

Mangrove plants comprise plants with similar ecological features that have enabled them to adapt to life between the sea and the land. Within a geographic region, different mangrove species share not only similar adaptations but also similar genetic structure patterns. Along the eastern coast of South America, there is a subdivision between the populations north and south of the continent's northeastern extremity. Here, we aimed to test for this north-south genetic structure in Rhizophora mangle, a dominant mangrove plant in the Western Hemisphere. Additionally, we aimed to study the relationships between R. mangle, R. racemosa, and R. × harrisonii and to test for evidence of hybridization and introgression. Our results confirmed the north-south genetic structure pattern in R. mangle and revealed a less abrupt genetic break in the northern population than those observed in Avicennia species, another dominant and widespread mangrove genus in the Western Hemisphere. These results are consistent with the role of oceanic currents influencing sea-dispersed plants and differences between Avicennia and Rhizophora propagules in longevity and establishment time. We also observed that introgression and hybridization are relevant biological processes in the northeastern coast of South America and that they are likely asymmetric toward R. mangle, suggesting that adaptation might be a process maintaining this hybrid zone.

Population genetic analysis of Giardia duodenalis: genetic diversity and haplotype sharing between clinical and environmental sources.

Giardia duodenalis is a flagellated intestinal protozoan responsible for infections in various hosts including humans and several wild and domestic animals. Few studies have correlated environmental contamination and clinical infections in the same region. The aim of this study was to compare groups of Giardia duodenalis from clinical and environmental sources through population genetic analyses to verify haplotype sharing and the degree of genetic similarity among populations from clinical and environmental sources in the metropolitan region of Campinas. The results showed high diversity of haplotypes and substantial genetic similarity between clinical and environmental groups of G. duodenalis. We demonstrated sharing of Giardia genotypes among the different populations studied. The comparison between veterinary and human sequences led us to identify new zoonotic genotypes, including human isolates from genetic assemblage C. The application of a population genetic analysis in epidemiological studies allows quantification of the degree of genetic similarity among populations of Giardia duodenalis from different sources of contamination. The genetic similarity of Giardia isolates among human, veterinary, and environmental groups reinforced the correlation between clinical and environmental isolates in this region, which is of great importance for public health.

Microsatellite loci for Urochloa decumbens (Stapf) R.D. Webster and cross-amplification in other Urochloa species.

BACKGROUND: Forage grasses of the African genus Urochloa (syn. Brachiaria) are the basis of Brazilian beef production, and there is a strong demand for high quality, productive and adapted forage plants. Among the approximately 100 species of the genus Urochloa, Urochloa decumbens is one of the most important tropical forage grasses used for pastures due to several of its agronomic attributes. However, the level of understanding of these attributes and the tools with which to control them at the genetic level are limited, mainly due to the apomixis and ploidy level of this species. In this context, the present study aimed to identify and characterize molecular microsatellite markers of U. decumbens and to evaluate their cross-amplification in other Urochloa species. FINDINGS: Microsatellite loci were isolated from a previously constructed enriched library from one U. decumbens genotype. Specific primers were designed for one hundred thirteen loci, and ninety-three primer pairs successfully amplified microsatellite regions, yielding an average of 4.93 alleles per locus. The polymorphism information content (PIC) values of these loci ranged from 0.26 to 0.85 (average 0.68), and the associated discriminating power (DP) values ranged from 0.22 to 0.97 (average 0.77). Cross-amplification studies demonstrated the potential transferability of these microsatellites to four other Urochloa species. Structure analysis revealed the existence of three distinct groups, providing evidence in the allelic pool that U. decumbens is closely related to Urochloa ruziziensis and Urochloa brizantha. The genetic distance values determined using Jaccard's coefficient ranged from 0.06 to 0.76. CONCLUSIONS: The microsatellite markers identified in this study are the first set of molecular markers for U. decumbens species. Their availability will facilitate understanding the genetics of this and other Urochloa species and breeding them, and will be useful for germplasm characterization, linkage mapping and marker-assisted selection.

In vitro Determination of Extracellular Proteins from Xylella fastidiosa.

The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3-30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.

The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles.

The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.

Genetic Structure and Molecular Diversity of Cacao Plants Established as Local Varieties for More than Two Centuries: The Genetic History of Cacao Plantations in Bahia, Brazil.

Bahia is the most important cacao-producing state in Brazil, which is currently the sixth-largest country worldwide to produce cacao seeds. In the eighteenth century, the Comum, Pará and Maranhão varieties of cacao were introduced into southern Bahia, and their descendants, which are called 'Bahian cacao' or local Bahian varieties, have been cultivated for over 200 years. Comum plants have been used to start plantations in African countries and extended as far as countries in South Asia and Oceania. In Brazil, two sets of clones selected from Bahian varieties and their mutants, the Agronomic Institute of East (SIAL) and Bahian Cacao Institute (SIC) series, represent the diversity of Bahian cacao in germplasm banks. Because the genetic diversity of Bahian varieties, which is essential for breeding programs, remains unknown, the objective of this work was to assess the genetic structure and diversity of local Bahian varieties collected from farms and germplasm banks. To this end, 30 simple sequence repeat (SSR) markers were used to genotype 279 cacao plants from germplasm and local farms. The results facilitated the identification of 219 cacao plants of Bahian origin, and 51 of these were SIAL or SIC clones. Bahian cacao showed low genetic diversity. It could be verified that SIC and SIAL clones do not represent the true diversity of Bahian cacao, with the greatest amount of diversity found in cacao trees on the farms. Thus, a core collection to aid in prioritizing the plants to be sampled for Bahian cacao diversity is suggested. These results provide information that can be used to conserve Bahian cacao plants and applied in breeding programs to obtain more productive Bahian cacao with superior quality and tolerance to major diseases in tropical cacao plantations worldwide.

VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as ß-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures.

Highly-sensitive and label-free indium phosphide biosensor for early phytopathogen diagnosis.

The development of highly-sensitive and label-free operating semiconductor-based, biomaterial detecting sensors has important applications in areas such as environmental science, biomedical research and medical diagnostics. In the present study, we developed an Indium Phosphide (InP) semiconductor-based resistive biosensor using the change of its electronic properties upon biomaterial adsorption as sensing element. To detect biomaterial at low concentrations, the procedure of functionalization and covalent biomolecule immobilization was also optimized to guarantee high molecule density and high reproducibility which are prerequisite for reliable results. The characterization, such as biomolecular conjugation efficiency, detection concentration limits, receptor:ligand specificity and concentration detection range was analyzed by using three different biological systems: i) synthetic dsDNA and two phytopathogenic diseases, ii) the severe CB-form of Citrus Tristeza Virus (CTV) and iii) Xylella fastidiosa, both causing great economic loss worldwide. The experimental results show a sensitivity of 1 pM for specific ssDNA detection and about 2 nM for the specific detection of surface proteins of CTV and X. fastidiosa phytopathogens. A brief comparison with other semiconductor based biosensors and other methodological approaches is discussed and confirms the high sensitivity and reproducibility of our InP based biosensor which could be suitable for reliable early infection diagnosis in environmental and life sciences.

A molecular linkage map for Drosophila mediopunctata confirms synteny with Drosophila melanogaster and suggests a region that controls the variation in the number of abdominal spots.

The classic approach to gene discovery relies on the construction of linkage maps. We report the first molecular-based linkage map for Drosophila mediopunctata, a neotropical species of the tripunctata group. Eight hundred F(2) individuals were genotyped at 49 microsatellite loci, resulting in a map that is ≈450 centimorgans long. Five linkage groups were detected, and the species' chromosomes were identified through cross-references to BLASTn searches and Müller elements. Strong synteny was observed when compared with the Drosophila melanogaster chromosome arms, but little conservation in the gene order was seen. The incorporation of morphological data corresponding to the number of central abdominal spots on the map was consistent with the expected location of a genomic region responsible for the phenotype on the second chromosome.

New microsatellite markers developed from Urochloa humidicola (Poaceae) and cross amplification in different Urochloa species.

BACKGROUND: Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species. FINDINGS: Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913. CONCLUSIONS: This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

Characterization of microsatellite loci in Himatanthus drasticus (Apocynaceae), a medicinal plant from the Brazilian savanna.

PREMISE OF THE STUDY: We developed a new set of microsatellite markers for studying the genome of the janaguba tree, Himatanthus drasticus (Mart.) Plumel, which is used in folk medicine in northeastern Brazil. These novel markers are being used to evaluate the effect of harvesting on the genetic structure and diversity of natural populations of this species. • METHODS AND RESULTS: Microsatellite loci were isolated from an enriched H. drasticus genomic library. Nine primer pairs successfully amplified polymorphic microsatellite regions, with an average of 8.5 alleles per locus. The average values of observed and expected heterozygosity were 0.456 and 0.601, respectively. • CONCLUSIONS: The microsatellite markers described here are valuable tools for population genetics studies of H. drasticus. The majority of the primers also amplified sequences in the genome of another species of the same genus. This new set of markers may be useful in designing a genetic conservation strategy and a sustainable management plan for the species.