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

BACKGROUND: Giardia 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 FINDINGS: For 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. CONCLUSIONS: There 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.

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.

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.

Species distribution and introgressive hybridization of two Avicennia species from the Western Hemisphere unveiled by phylogeographic patterns.

BACKGROUND: Mangrove plants grow in the intertidal zone in tropical and subtropical regions worldwide. The global latitudinal distribution of the mangrove is mainly influenced by climatic and oceanographic features. Because of current climate changes, poleward range expansions have been reported for the major biogeographic regions of mangrove forests in the Western and Eastern Hemispheres. There is evidence that mangrove forests also responded similarly after the last glaciation by expanding their ranges. In this context, the use of genetic tools is an informative approach for understanding how historical processes and factors impact the distribution of mangrove species. We investigated the phylogeographic patterns of two Avicennia species, A. germinans and A. schaueriana, from the Western Hemisphere using nuclear and chloroplast DNA markers. RESULTS: Our results indicate that, although Avicennia bicolor, A. germinans and A. schaueriana are independent lineages, hybridization between A. schaueriana and A. germinans is a relevant evolutionary process. Our findings also reinforce the role of long-distance dispersal in widespread mangrove species such as A. germinans, for which we observed signs of transatlantic dispersal, a process that has, most likely, contributed to the breadth of the distribution of A. germinans. However, along the southern coast of South America, A. schaueriana is the only representative of the genus. The distribution patterns of A. germinans and A. schaueriana are explained by their different responses to past climate changes and by the unequal historical effectiveness of relative gene flow by propagules and pollen. CONCLUSIONS: We observed that A. bicolor, A. germinans and A. schaueriana are three evolutionary lineages that present historical and ongoing hybridization on the American continent. We also inferred a new evidence of transatlantic dispersal for A. germinans, which may have contributed to its widespread distribution. Despite the generally wider distribution of A. germinans, only A. schaueriana is found in southern South America, which may be explained by the different demographic histories of these two species and the larger proportion of gene flow produced by propagules rather than pollen in A. schaueriana. These results highlight that these species responded in different ways to past events, indicating that such differences may also occur in the currently changing world.

Multiple-geographic-scale genetic structure of two mangrove tree species: the roles of mating system, hybridization, limited dispersal and extrinsic factors.

Mangrove plants comprise a unique group of organisms that grow within the intertidal zones of tropical and subtropical regions and whose distributions are influenced by both biotic and abiotic factors. To understand how these extrinsic and intrinsic processes influence a more fundamental level of the biological hierarchy of mangroves, we studied the genetic diversity of two Neotropical mangrove trees, Avicenniagerminans and A. schaueriana, using microsatellites markers. As reported for other sea-dispersed species, there was a strong differentiation between A. germinans and A. schaueriana populations sampled north and south of the northeastern extremity of South America, likely due to the influence of marine superficial currents. Moreover, we observed fine-scale genetic structures even when no obvious physical barriers were present, indicating pollen and propagule dispersal limitation, which could be explained by isolation-by-distance coupled with mating system differences. We report the first evidence of ongoing hybridization between Avicennia species and that these hybrids are fertile, although this interspecific crossing has not contributed to an increase in the genetic diversity the populations where A. germinans and A. schaueriana hybridize. These findings highlight the complex interplay between intrinsic and extrinsic factors that shape the distribution of the genetic diversity in these sea-dispersed colonizer species.

Molecular genetic variability of commercial and wild accessions of passion fruit (Passiflora spp.) targeting ex situ conservation and breeding.

Passiflora species are distributed throughout Latin America, and Brazil and Colombia serve as the centers of diversity for this genus. We performed cross-species amplification to evaluate 109 microsatellite loci in 14 Passiflora species and estimated the diversity and genetic structure of Passiflora cincinnata, Passiflora setaceae and Passiflora edulis. A total of 127 accessions, including 85 accessions of P. edulis, a commercial species, and 42 accessions of 13 wild species, were examined. The cross-species amplification was effective for obtaining microsatellite loci (average cross-amplification of 70%). The average number of alleles per locus (five) was relatively low, and the average diversity ranged from 0.52 in P. cincinnata to 0.32 in P. setacea. The Bayesian analyses indicated that the P. cincinnata and P. setacea accessions were distributed into two groups, and the P. edulis accessions were distributed into five groups. Private alleles were identified, and suggestions for core collections are presented. Further collections are necessary, and the information generated may be useful for breeding and conservation.

New microsatellite markers for wild and commercial species of Passiflora (Passifloraceae) and cross-amplification.

PREMISE OF THE STUDY: We developed the first microsatellites for Passiflora setacea and characterized new sets of markers for P. edulis and P. cincinnata, enabling further genetic diversity studies to support the conservation and breeding of passion fruit species. • METHODS AND RESULTS: We developed 69 microsatellite markers and, in conjunction with assessments of cross-amplification using primers available from the literature, present 43 new polymorphic microsatellite loci for three species of Passiflora. The mean number of alleles per locus was 3.1, and the mean values of the expected and observed levels of heterozygosity were 0.406 and 0.322, respectively. • CONCLUSIONS: These microsatellite markers will be valuable tools for investigating the genetic diversity and population structure of wild and commercial species of passion fruit (Passiflora spp.) and may be useful for developing conservation and improvement strategies by contributing to the understanding of the mating system and hybridization within the genus.

Development of microsatellite markers for Pimenta pseudocaryophyllus (Myrtaceae), a wild South American species.

PREMISE OF THE STUDY: We developed the first set of microsatellite markers for Pimenta pseudocaryophyllus to support further studies on genetic diversity and to inform conservation strategies. METHODS AND RESULTS: The microsatellite-enriched library approach was used to isolate and characterize 12 new molecular markers. It was possible to detect 11 polymorphic microsatellite loci and one monomorphic locus. The polymorphism information content ranged from 0.317 to 0.869. CONCLUSIONS: These molecular markers will be valuable tools to aid in understanding the biology of P. pseudocaryophyllus and to detect ongoing consequences of its exploitation, in the context of conservation genetics.

Development and characterization of microsatellite markers for the wild South American Passiflora cincinnata (Passifloraceae).

PREMISE OF THE STUDY: We developed the first set of microsatellite markers for the passion fruit, Passiflora cincinnata, to provide tools for further study of its genetic diversity and to support current conservation and genetic studies. METHODS AND RESULTS: We used a microsatellite-enriched library approach to isolate and characterize 25 new molecular markers. Seven primer pairs successfully amplified polymorphic microsatellite regions, with an average of five alleles per locus. The mean values of expected and observed levels of heterozygosity were 0.516 and 0.525, respectively. CONCLUSIONS: The microsatellite markers identified in this study may be valuable tools for population genetic studies, and this set of markers also may be useful in the design of a genetic conservation strategy, mating system, and hybridization studies.

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.

Microsatellites for the mangrove tree Avicennia germinans (Acanthaceae): Tools for hybridization and mating system studies.

PREMISE OF THE STUDY: We developed a new set of microsatellite markers for the black mangrove Avicennia germinans, to provide new informative tools for further studies of the mating system, interspecific hybridization, and population genetics. • METHODS AND RESULTS: We used the microsatellite-enriched library approach to isolate and characterize 25 new primer pairs. Sixteen of them are polymorphic, showing a variable degree of variation in A. germinans, while nine were monomorphic in the samples examined. Eight exhibited private alleles in A. schaueriana. • CONCLUSIONS: These results indicate that these new microsatellite markers will be useful molecular tools for further studies of A. germinans and A. schaueriana population genetics, mating systems, and hybridization.