Development and characterization of microsatellite loci for Ocotea species (Lauraceae) threatened with extinction

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes13351385142COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESsem informaçã

Tapping latex and alleles? The impacts of latex and bark harvesting on the genetic diversity of Himatanthus drasticus (Apocynaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The importance of genetic variation for maintaining biological diversity and evolutionary processes is widely accepted; however, limited action has been taken to protect genetic diversity in natural populations. Until recently, it was assumed that the exploitation of non-timber forest products, such as fruits, barks and exudates, had low environmental impact, but recognition that the harvesting of these plants can affect several biological levels, including genetic diversity and structure, has been growing. Nevertheless, the effect of harvesting non-timber forest products on the genetic diversity of the harvested species is poorly understood. The objective of this work is to assess the impact of harvesting on the genetic diversity of Himatanthus drasticus (Apocynaceae), a tree of the Brazilian Savanna (Cerrado) that is highly exploited for its medicinal latex. We tested the hypothesis that harvesting can cause a trade-off between survival and reproduction in H. drasticus, reducing the effective population size as well as the genetic diversity in highly exploited populations. The study was conducted in the Araripe National Forest, a preserved Cerrado area in Ceara State, Brazil. We sampled six populations that included two different levels of harvesting (high-harvest and low-harvest) and characterized the genetic diversity and structure in two groups (adults and seedlings) using microsatellite markers. We verified high levels of polymorphism in H. drasticus; however, significant levels of inbreeding in all studied populations were also observed. We found a higher degree of genetic structure in the seedling group when compared with the adult group, a difference that was most likely due to seed rain deposited in nearby locations. No differences were detected in adult genetic diversity levels between populations under different harvesting intensities. However, seedling allelic richness was significantly lower in the high-harvested populations, indicating allelic losses that may be associated with the exploitation of bark and latex. Evidence of genetic loss due to overharvesting was supported by significant bottleneck signatures observed in the seedling group of two out of three high-harvest populations. The establishment of a conservation strategy is imperative to avoid future genetic losses and ensure the long-term viability of H. drasticus populations. The information obtained in this study has been integrated with ecological information about the effects of latex tapping on phenology, demography and biomass recovery of exploited populations, with the goal of proposing a sustainable management plan for the species. (C) 2013 Elsevier B.V. All rights reserved.310434441Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq [472127/2008-0]FAPESP [2008/08737-4

Characterization Of 10 Microsatellite Loci For Bathysa Australis (rubiaceae).

• Bathysa australis is a common subcanopy tree from the Atlantic Forest that is pollinated by bees and wasps and produces autochoric seeds. This species exhibits great phenotypic plasticity along the elevational gradient of Serra do Mar in southeastern Brazil. We expect to assess the genetic diversity and gene flow between populations of this species along the elevational gradient. • We developed a microsatellite-enriched genomic library for B. australis, and 10 microsatellite loci were successfully amplified, ranging from one to 13 alleles per locus. The observed and expected heterozygosities ranged from 0.333 to 0.900 (average: 0.629) and 0.564 to 0.900 (average: 0.742), respectively. • These are the first microsatellite markers developed for the genus Bathysa and may be useful in other species of the Condamineeae tribe. These primers will be an important tool for studies of population ecology and conservation genetics.

First Report of Colletotrichum musicola Causing Soybean Anthracnose in Brazil

Soybean (Glycine max L.) is one of the most important crops worldwide as a source of protein-rich foods and animal feeds. Anthracnose, one of the major limiting factors to soybean production (Dias et al. 2016), is caused by species such as Colletotrichum truncatum, C. sojae, and C. plurivorum (Damm et al. 2009, 2019). In December 2016 and 2017, soybean plants of cultivars Monsoy 8768 and Pioneer y-70 with typical symptoms of anthracnose (necrotic and irregular brown lesions on stems, leaves, and pods) were collected in Mato Grosso, Brazil. Commercial fields sampled showed 10 to 15% incidence of anthracnose in 1 ha in each sampled area. In total, 10 different geographic locations were sampled. Colletotrichum strains were isolated and cultured on potato dextrose agar at 25°C with a 12-h light photoperiod from surface-disinfected (70% alcohol followed by 0.5% sodium hypochlorite) plant tissues. Among others, three single-spore isolates (LFN0048 from Sinop, LFN0074 and LFN0090 from Lucas do Rio Verde) showed different morphology; isolates LFN0048 and LFN0074 were selected for further characterization. Total DNA was extracted and partial glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), and β-tubulin (TUB2) genes were amplified and sequenced. The sequences were deposited in GenBank (accession numbers MN604249 and MK163893 for HIS3, MN604248 and MK142674 for GAPDH, and MN604250 and MK142675 for TUB) and were compared with most similar reference sequences of Colletotrichum (Damm et al. 2019). Both isolates clustered with Colletotrichum musicola epitype (CBS 132885), showing 100 and 98.5% similarity in GAPDH, 99.5 and 98.9% in HIS3, and 99.2% in TUB2. On PDA, colonies showed dark-gray aerial mycelium with entire margins, reverse violaceous-black. Conidia and ascospore size and shape match those previously described by Damm et al. (2019): 12.12 to 15.86 × 4.93 to 6.95 µm and 15.5 to 19.34 × 5 to 7.84 µm, respectively (n = 100). Appressoria (n = 50) were single or in loose groups, violaceous-black with predominant obovoid, truncated, and cylindrical shapes, with smooth, undulate, or lobate margin, and 9.25 to 29.79 × 7.22 to 21.06 µm. Perithecia, paraphyses; and unitunicate eight-spored asci were also observed. Asci were cylindrical to clavate, smooth-walled, and 48.12 to 68.78 × 9.59 to 14.47 µm (n = 50). Soybean anthracnose is seed-borne (Dias et al. 2018; Rogério et al. 2017); therefore, pathogenicity tests were carried out on pregerminated seeds. Five seeds of Brasmax 8579 cultivar were inoculated with 10 µl of a conidial suspension (106 conidia/ml) that was placed in the emerging radicle, and five mock-inoculated seeds were used as a control. Seedlings were planted in vermiculite and incubated at 25°C with a 12-h photoperiod. After 7 days, inoculated plants showed necrotic lesions on the cotyledons, leaflets, and hypocotyl, whereas control plants remained asymptomatic. The experiment was repeated three times. C. musicola was reisolated from the symptomatic tissues, and the identity was confirmed by morphology and multilocus phylogeny. Until now, C. musicola has been reported to be associated with Musa sp. (Damm et al. 2019) and Colocasia esculenta (Vásquez-López et al. 2019) in Mexico, and with Phaseolus lunatus in Brazil (Cavalcante et al. 2018). To our knowledge, this is the first report of C. musicola joining a group of new and emergent species of Colletotrichum causing anthracnose in soybean-producing regions around the world

Molecular genotyping, diversity studies and high-resolution molecular markers unveiled by microsatellites in Giardia duodenalis.

BackgroundGiardia duodenalis (synonyms G. lamblia and G. intestinalis) is an enteric protozoan parasite of a wide range of mammalian hosts, including humans and various domestic and wild animals. There is considerable genetic variability in G. duodenalis and isolates of this parasite have been divided into eight genetic assemblages. Microsatellites markers can be used to discriminate isolates with a high level of sensitivity. This study was conducted to identify and characterize genomic microsatellites (simple sequence repeats-SSRs), sequences of one- to six-nucleotide motifs repeated in tandem, present in the available genomes of G. duodenalis and to develop new markers that can serve as a tool for detection and for characterizing the genetic diversity of this parasite.Methodology/ principal findingsFor each genetic assemblage, polymorphism levels for the microsatellite markers were evaluated. After performing the analysis using the MISA and SciRoKo software, 1,853 simple sequence repeats (SSRs) were identified. In all the genomes, trinucleotide repeats were the most common class followed by tetranucleotide. Many of the SSR loci are assemblage-specific, and 36 SSR loci shared among all the genomes were identified. Together with hypothetical proteins, variant-specific surface proteins represented nearly half of the annotated SSR loci. The results regarding the most common repeat among the SSRs led us to infer that positive selection occurred to avoid frameshift mutations. Additionally, based on inter- and intra-genetic assemblages polymorphism analyses, we unveiled previously undetected genetic variation, indicating that the microsatellite markers we developed are useful molecular tools for epidemiological inferences based on population genetics patterns and processes.ConclusionsThere is increasing demand for the development of new molecular markers and for the characterization of pathogens at a higher resolution level. In this study, we present 60 G. duodenalis microsatellites markers that exhibited high polymerase chain reaction (PCR) amplification efficiency among the different genetic assemblages. Twenty of these markers presented nucleotide sequence polymorphisms and may be used as a genotyping tool. The monomorphic markers can be used for the detection of the parasite at the species and genetic assemblage level. These polymorphic markers revealed a genetic diversity that was previously undetectable, thus they can be considered valuable molecular tools for high resolution markers in future studies investigating Giardia and may also be used for epidemiological inferences based on populations genetics patterns and processes

Development and characterization of microsatellite loci for Ocotea species (Lauraceae) threatened with extinction

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes

Development And Characterization Of Microsatellite Loci For Ocotea Species (lauraceae) Threatened With Extinction

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 × 10-24 to 7.7 × 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes. © FUNPEC-RP.13351385142Araújo, P.A.M., Fichas dendrológicas comerciais e industriais brasileiras: Imbuia (1948) Anu. Bras. Econ. Flor., 1, pp. 348-352Billotte, N., Lagoda, P.J.L., Risterucci, A.M., Baurens, F.C., Microsatellite-enriched libraries: Applied methodology for the development of SSR markers in tropical crops (1999) Fruits, 54, pp. 277-288Carvalho, P.E.R., (1994) Espécies Florestais Brasileiras: Recomendações Silviculturais, Potencialidades e Uso da Madeira., , EMBRAPA-CNPF, BrasíliaDoyle, J.J., Doyle, J.L., A rapid DNA isolation procedure for a small amount of fresh leaf tissue (1987) Phytochem. Bull., 19, pp. 11-15Goudet, J., (2001) FSTAT, A Program to Estimate and Test Gene Diversities and Fixation Indices (version 2.9.3)., , http://www2.unil.ch/popgen/softwares/fstat.htm, Available at Accessed August 2012Klein, R., (1980) Ecologia da Flora e Vegetação do Vale do Itajaí., p. 389. , SellowiaMartins, W.S., Lucas, D.C., Neves, K.F., Bertioli, D.J., WebSat-a web software for microsatellite marker development (2009) Bioinformation, 3, pp. 282-283(2008) Instrução Normativa n°.6 de 23/09/2008., , MMA (Ministério do Meio Ambiente)Peakall, R., Smouse, P.E., GenAlEx 6: Genetic analysis in Excel. Population genetic software for teaching and research (2006) Mol. Ecol. Notes, 6, pp. 288-295Reitz, R., Klein, R.M., Reis, A., (1978) Projeto Madeira de Santa Catarina., pp. 28-30. , SellowiaRohwer, J.G., Toward a phylogenetic classification of the Lauraceae: Evidence from matK sequences (2000) Syst. Bot., 25, pp. 60-71Rozen, S., Skaletsky, H.J., Primer3: Bioinformatics Methods and Protocols (2000) Methods in Molecular Biology., pp. 365-386. , http://http://frodo.wi.mit.edu/, In: (Krawetz S and Misener S, eds.). Humana Press, New Jersey, Available at Accessed August 2012Van Oosterhout, C., Hutchinson, W.F., Wills, D.P.M., Shipley, P., Micro-Checker: Software for identifying and correcting genotyping erros in microsatellite data (2004) Mol. Ecol. Notes, 4, pp. 535-538Varty, N., (1998) Ocotea pretiosa, , www.iucnredlist.org, IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. Available at. Accessed April 17, 2012Varty, N., Guadagnin, D.L., (1998) Ocotea catharinensis, , www.iucnredlist.org, (1998a). IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. Available at Accessed April 17, 2012Varty, N., Guadagnin, D.L., (1998) Ocotea porosa, , www.iucnredlist.org, (1998b). IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. Available at Accessed April 17, 201

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 health62CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP577032/2008-9; 304914/2010-02008/52197-4; 2008/52197-4; 2011/50413-