Molecular data reveal a complex population genetic structure for Psalidodon scabripinnis (Teleostei: Characidae) in the Atlantic Rainforest, Brazil.

Recently renamed, Psalidodon scabripinnis populations of Serra da Mantiqueira, previously known as Astyanax scabripinnis have been deeply studied in the last years. These populations are small and isolated and occur very close to the watershed between Paraíba do Sul River basin and Upper Paraná River basin, in Serra da Mantiqueira region in the Atlantic Rainforest. These conditions arouse the interest in knowing theor genetic conservation status and how they responded to the separation between the two rivers basins. Therefore, we accessed the genetic diversity of five P. scabripinnis populations of this region with microsatellites and mitochondrial data. The results showed a complex structure pattern that doesn't match the simple basin separation and a reasonably conservation status when compared with other populations of the same family or with similar natural history.

Evaluation of a panel of microsatellite markers to study their applications in Serrapinnus notomelas and to reveal the genetic diversity in Hyphessobrycon eques.

Jewel tetra (Hyphessobrycon eques) is a freshwater fish found in several rivers and basins in South America. The present study is the first study to create a panel of microsatellite markers for detecting genetic diversity in H. eques and evaluating the application of these markers in Serrapinnus notomelas. In total, 44 individuals were genotyped from the natural (WIL, n = 20) and stock in captivity (CAP, n = 24) population. Moreover, 19 microsatellite markers were obtained, of which only 8 loci presented a high degree polymorphism. In total, 45 alleles were detected, ranging from 126 bp (Hype2G2) to 420 bp (Hype2E2). The Hardy-Weinberg equilibrium (p < 0.05) revealed significant difference in one locus in WIL (Hype1G4) and three loci in CAP (Hype1F4, Hype2C3, and Hype2G2). Null alleles (p < 0.05) were present in only one locus (Hype1G4). The WIL and CAP populations revealed high genetic diversity during FST analysis. The cross-amplification test for S. notomelas revealed that only two loci (Hype2C3 and Hype2G2B) presented satisfactory transferability results. The developed microsatellite primers will be useful in studying the genetic diversity and population structure of H. eques in wild populations and fish farms in the Brazilian and other South American basins.

Phenotypic, molecular and pathogenic characterization of Colletotrichum scovillei infecting Capsicum species in Rio de Janeiro, Brazil.

Anthracnose is a disease caused by Colletotrichum spp., one of the world's most damaging sweet and chili pepper pathogens, especially in tropical and subtropical regions. In the state of Rio de Janeiro, anthracnose is one of the main obstacles for pepper crops. However, to date no research has focused on the identification and characterization of the pathogen, which is fundamental to understand the scope of the disease in the state. Thus, the correct identification of the fungal species and pathogenicity studies can provide important support for disease management and control, apart from identifying possible resistance sources for exploitation in peppers breeding programs. In this study, 11 Colletotrichum isolates were collected from peppers with typical symptoms in the Rio de Janeiro state. These isolates were characterized based on morpho-cultural characteristics and sequencing data from five regions (ITS, ACT, CAL, ß-TUB and GAPDH), and the genetic variability was estimated by AFLP markers. Simultaneously, microscopy images of the colonization by the fungal species on unripe Capsicum annuum fruits were taken. Pathogenicity was tested and resistance sources were sought by means of infection of ripe and unripe fruits of 50 Capsicum baccatum accessions. The resulting data showed that all isolates belong to Colletotrichum scovillei specie. About the pathogenicity of Capsicum baccatum, differentiated, stage-specific responses, with higher resistance of ripe fruits were recorded. In addition, four possible sources of Colletotrichum scovillei resistance were detected among the tested accessions. The combination of these data can contribute to future studies on the interaction of Colletotrichum scovillei-Capsicum spp., a research line that is still unexploited in the main areas of this anthracnose fungus.

Structure and genetic variability of golden mussel (Limnoperna fortunei) populations from Brazilian reservoirs.

The golden mussel, Limnoperna fortunei a highly invasive species in Brazil, has generated productive, economical, and biological impacts. To evaluate genetic structure and variability of L. fortunei populations present in fish farms in the reservoirs of Canoas I (CANFF), Rosana (ROSFF), and Capivara (CAPFF) (Paranapanema River, Paraná, Brazil), eight microsatellite loci were amplified. Five of those eight loci resulted in 38 alleles. The observed heterozygosity (Ho) was lower than the expected heterozygosity (He) in all populations, with a deviation from the Hardy-Weinberg equilibrium (HWE). The average value for the inbreeding coefficient (Fis) was positive and significative for all populations. There was higher genetic variability within populations than among them. The fixation index (Fst) showed a small genetic variability among these populations. The occurrence of gene flow was identified in all populations, along with the lack of a recent bottleneck effect. The clustering analysis yielded K = 2, with genetic similarity between the three populations. The results demonstrate low genetic structure and suggest a founding population with greater genetic variability (ROSFF). Our data point to the possible dispersal of L. fortunei aided by anthropic factors in the upstream direction. It was concluded that the three populations presented a unique genetic pool for Paranapanema River, with occurrence of gene flow.

Distinctness of Brazilian common bean cultivars with carioca and black grain by means of morphoagronomic and molecular descriptors.

Common bean (Phaseolus vulgaris L.) is one of the most important legumes for human consumption and is a staple food in the diet of the population of some countries of Latin America, Africa and Asia. The distinction between cultivars is based predominantly on morphological descriptors, which proved inefficient for the differentiation of some cultivars. This study had the objective of describing, distinguishing and evaluating the agronomic potential of 39 common bean cultivars of the carioca and black grain groups registered for cultivation in Brazil, based on 49 morphoagronomic descriptors and microsatellite (simple sequence repeat -SSR) markers. The morphoagronomic traits of each commercial group were characterized in four environments. Thirty-seven SSR markers were used for the molecular description. The morphological data, analyzed by the Shanonon-Weaver index, detected low variability among cultivars for qualitative data. On the other hand, the estimates of variance analysis, relative importance of the traits and hierarchical grouping analysis applied to the quantitative variables showed that the descriptors related to plant morphology were the most important for the carioca group, and those related to seed morphology were determining for the black group. The genetic parameters estimated for SSR markers by hierarchical and Bayesian cluster analysis identified 116 alleles, with 33 and 30 polymorphic loci and 24 and 22 private alleles for the carioca and black groups, respectively. The combined use of morphoagronomic and molecular descriptors improves the distinguishability of cultivars, contributing in a more efficient way to breeding and to the protection of cultivars.

Genetic variability of three natural populations of Maytenus aquifolium (Celesteraceae) from Telêmaco Borba, Paraná, Brazil

Three populations of Maytenus aquifolium from Monte Alegre farm, Telemaco Borba county, Paraná, Brazil were analyzed by RAPD markers. A total of 13 primers were applied wich yielded 283 well amplified markers in all the studied populations (Mortandade, Vila Preta and Trinita), producing different values of gene diversity and polymorphic loci. The analysis of molecular variance (AMOVA) indicated that 21.77 percent of the genetic variation was among the population. Pairwise F ST analysis showed that the most divergent populations were closer geographically, demonstrating that other factors such as different soil types could explain this variation. Bayesian analysis for K number of clusters and the Principal Coordinate indicated that these three populations were highly structured, corroborating the high values found for the F ST and indicating that for conservation purposes all populations should be maintained.

Três populações de Maytenus aquifolium coletadas na fazenda Monte Alegre, localizada no município de Telêmaco Borba, Paraná, Brasil foram analisadas por marcadores de RAPD. Treze primers de RAPD foram amplificados produzindo 283 bandas com alta capacidade de repetição nas três populações estudadas (Mortandade, Trinita e Vila Preta), com as quais valores diferentes de diversidade gênica e locos poplimórficos foram calculados. Análise da variância molecular (AMOVA) mostrou que 21,77 por cento da variabilidade genética é encontrada entre populações. Análise do Fst entre pares de populações mostrou que as mais divergentes são geograficamente mais próximas, demonstrando que outros fatores tais como tipos distintos de solos podem explicar estas variações. Análise Bayesiana para número K de clusters e a Análise da Coordenada Principal mostraram que estas três populações estão altamente estruturadas, corroborando os altos valores encontrados para o Fst e indicando que para propósitos de conservação todas as populações devem ser mantidas.

Genetic analysis of species in the Genus Catasetum (ORCHIDACEAE) using RAPD Markers

In this work, RAPD molecular markers were used to access the genetic variability and to study the inter and intraespecifc relationship in a group of 37 species, including 56 individuals. A total of 15 RAPD primers were selected for DNA amplification. From a total of 221 bands analyzed, 209 (95 percent) were polymorphics. The level of interespecifc genetic similarity ranged from 37 percent between Catasetum complanatum and Catasetum laminatum to 83 percent between Catasetum triodon and Catasetum uncatum. The intraspecifc genetic similarity varied 88 percent for the individuals of Catasetum triodon to 93 percent between the individuals of Catasetum atratum and Catasetum macrocarpum. These results would contribute to understand the genetic relationship in Catasetum, to define the strategies to establish a germplasm core collection for the genus and to provide support for breeding programs.

Neste trabalho, marcadores moleculares de RAPD foram utilizados para acessar a variabilidade genética e estudar as relações interespecíficas e intraespecífica em um grupo de 37 espécies, compreendendo 56 plantas individuais. Um total de 15 primers foram selecionados para amplificação do DNA. De um total de 221 bandas analisadas, 209 (95 por cento) foram polimórficas. O nível de similaridade genética interespecífica variou de 37 por cento entre Catasetum complanatum e Catasetum laminatums a 83 por cento entre Catasetum triodon e Catasetum uncatum. A similaridade genética intraespecífica variou de 88 por cento entre os indivíduos de Catasetum triodon a 93 por cento entre os indivíduos de Catasetum atratum e Catasetum macrocarpum. Os resultados deste trabalho contribuem para o entendimento das relações interespecíficas no gênero Catasetum, para definir estratégias para o estabelecimento de um banco de germoplasma e para dar suporte a programas de melhoramento.

AFLP and breeding system studies indicate vicariance origin for scattered populations and enigmatic low fecundity in the Moroccan endemic Hypochaeris angustifolia (Asteraceae), sister taxon to all of the South American Hypochaeris species.

We used Amplified Fragment Length Polymorphism markers (AFLP) and breeding system studies to investigate the population structure and reproductive biology of Hypochaeris angustifolia (Asteraceae: Cichorieae). This species is endemic to altiplanos of the Atlas Mountains (Morocco) where it occurs in scattered populations, and it is the sister species to c. 40 species of this genus in South America. PCoA, NJ, and Bayesian clustering, revealed that the populations are very isolated whilst AFLP parameters show that almost all populations have marked genetic divergence. We contend that these features are more in accord with a vicariance origin for the scattered populations of H. angustifolia, rather than establishment by long-distance dispersal. The breeding system studies revealed that H. angustifolia is a self-incompatible species, with low fecundity in natural and in experimental crosses, probably due to a low frequency of compatible phenotypes within and between the populations.

Comparative analysis of genetic diversity among the maize inbred lines (Zea mays L.) obtained by RAPD and SSR markers

The RAPD and SSR markers were used to compare the genetic diversity among the 16 maize inbred lines. Twenty-two primers were used in the RAPD reactions, resulting in the amplification of 265 fragments, while 16 pairs of SSR primers resulted in 75 fragments. The similarity based on Dice coefficient for the RAPD ranged from 53 to 84 percent and for the SSR from 11 to 82 percent. The dendrogram obtained by the RAPD showed five groups, while dendrogram obtained by the SSR showed three groups and one isolated line. The association constructed from the markers and the principal coordinate’s analysis separated lines into two groups according to endosperm color, either orange or yellow. The RAPD were effective to validate pedigree data, while the SSR were effective to recognize the differences between the quantitative characters. Because they assess the distinct regions of the genome, the selection of one or other marker would depend on the characteristics of the material used and the objectives of the project.

RAPD e SSR foram utlizados para comparar a diversidade genética entre 16 linhagens de milho. Nas reações de RAPD foram utlizados 22 primers que resultaram na amplificação de 265 fragmentos, enquanto que 16 pares de primes de SSR resultaram em 75 fragmentos. A similaridade baseada no coeficiente de Dice variou de 53 por cento a 84 por cento para o RAPD; para o SSR variou de 11 por cento a 82 por cento. O dendrograma obtido a partir do RAPD mostrou 5 grupos enquanto que o dendrograma obtido a partir do SSR mostrou 3 grupos e uma linhagem isolada. A associação construída a partir dos marcadores e a análise de coordenadas principais separaram as linhagens em dois grupos de acordo coloração de endosperma alaranjado ou amarelo, os marcadores RAPD foram eficientes para a validação dos dados de pedigree enquanto os de microssatélites para reconhecerem diferenças entre caracteres quantitativos. Por acessarem regiões distintas do genoma a escolha de um ou outro marcador vai depender das características do material utilizado e dos objetivos do trabalho.

Genetic diversity among forty coffee varieties assessed by RAPD markers associated with restriction digestion

A variabilidade genética de 40 acessos de cafeeiros de fenótipo arabica foi obtida usando a técnica de RAPD associada a uma digestão prévia do DNA genômico com endonucleases. A variabilidade genética e a relação entre os accessos foram inicialmente avaliadas pela amplificação de 195 primers. Para incrementar a eficiência na detecção de polimorfismo, o DNA genômico de cada acessos foi submetido a digestão com endonucleases antes da PCR. Um total de 24 primers combinados com restrição do DNA gerou 318 bandas, das quais 266 (83,65%) foram polimórficas. A associação entre os 40 acessos foi estimada pelo método de clusters UPGMA, sendo os acessos agrupados de acordo com seu pedigree e aspectos agronômicos. Os resultados mostraram que o uso de enzimas de restrição antes da reação de amplificação pode ser considerada uma ferramenta eficiente para incrementar o número de bandas informativas, possibilitando a diferenciação entre os 40 acessos de C. arabica.

Genetic variability of pre and post-fragmentation cohorts of Aspidosperma polyneuron Muell. Arg. (Apocynaceae)

O método de RAPD foi usado para acessar a variabilidade genética em Aspisdosperma polyneuron, uma árvore tropical de vida longa e idade reprodutiva tardia, e muito importante na Floresta Atlântica. Amostras foram coletadas em seis fragmentos florestais na região da cidade de Londrina (Sul do Brasil), uma paisagem fragmentada na década de 30. O perfil de RAPD foi analisado em adultos (pré-fragmentação, >300 anos) e plântulas (pós-fragmentação, <<50 anos). Os resultados mostram uma queda no polimorfismo genético em gerações pós-fragmentação nos pequenos fragmentos e alta diversidade genética dentro das populações. A distribuição da diversidade genética sugere o estabelecimento dos fragmentos como reservas protegidas, e a transferência de plântulas entre os fragmentos para a conservação de A. polyneuron.

Chromosomal organization and phylogenetic relationships in Hypochaeris species (Asteraceae) from Brazil

The association of cytogenetic and molecular techniques has contributed to the analysis of chromosome organization and phylogeny in plants. The fluorochrome GC-specific CMA3, fluorescent in situ hybridization (FISH) and RAPD (Random Amplified Polymorphic DNA) markers were used to investigate chromosome structure and genetic relationships in Hypochaeris (Asteraceae). Seven species native to South America, and two species introduced from Europe (H. glabra and Hypochaeris sp) were studied. FISH with rDNA probes identified one or two loci of 18S-5.8S-25S rDNA in the South American Hypochaeris species and one locus in the European species. Only one 5S rDNA locus was seen in all species studied. Blocks of GC-rich heterochromatin (CMA-positive bands) associated to 18S-5.8S-25SrDNA loci were detected in all species investigated. Co-location of 5S rDNA and CMA bands was also observed, except for three South American species and Hypochaeris sp. In two South American species, additional CMA bands not related to rDNA were observed on the long arm of chromosome 2, near to the centromere. Hypochaeris glabra exhibited additional CMA-positive signals distributed at pericentromeric regions, on the short arms of all chromosomes. A total of 122 RAPD markers were used to determine the genetic relationships among species. The level of polymorphism was very high, revealing two genetic groups comprising the South American and the European species, thus supporting a previous hypothesis of monophyly of the South American Hypochaeris species. The coefficients of genetic similarity between European and South American species were 0.35, on average. Polymorphism was also high within the two groups. The genetic associations observed with RAPD markers were consistent with chromosome characteristics. Species carrying similar distribution of 45S rDNA loci and CMA-positive signals were included in the same group revealed by RAPDs. Cytogenetic and molecular data support the view that not only chromosome rearrangements, but also changes in DNA sequence took place during the diversification of the South American Hypochaeris species.

Genetic polymorphism among 14 elite Coffea arabica L. cultivars using RAPD markers associated with restriction digestion

Knowledge of the genetic variability among genotypes is important for the transfer of useful genes and to maximize the use of available germplasm resources. This study was carried out to assess the genetic variability of 14 elite Coffea arabica cultivars using random amplified polymorphic DNA (RAPD) associated with a prior digestion of genomic DNA with restriction endonucleases. The accessions were obtained from the Coffea collection maintained at the Instituto Agronomico do Parana (IAPAR), located in Londrina, Parana, Brazil. Twenty-four informative RAPD primers, used in association with restriction enzymes, yielded 330 reproducible and scorable DNA bands, of which 224 (68 per cent) were polymorphic. The amplified products were used to estimate the genetic variability using Dice's similarity coefficient. The data matrix was converted to a dendrogram and a three-dimensional plot using principal coordinate analysis. The accessions studied were separated into clusters in a manner that was consistent with the known pedigree. The associations obtained in the dendrogram and in the principal coordinate analysis plot suggest the probable origin of the Kattimor cultivar. The RAPD technique associated with restriction digestion was proved to be a useful tool for genetic characterization of C. arabica genotypes making an important contribution to the application of molecular markers to coffee breeding

Assessment of genetic variability within and among coffee progenies and cultivars using RAPD markers

The RAPD technique associated with restriction digestion of genomic DNA was used to assess the genetic variability within and among nine populations of Coffea arabica, including six progenies belonging to the Sarchimor germplasm, the progeny PR 77054-40-10 (Catuaí Vermelho IAC 81 x Icatu), and two commercial cultivars (IAPAR 59 and Catuaí Vermelho IAC-81). These populations were evaluated using analysis of molecular variance (AMOVA), genetic similarity among progenies, and percentage of polymorphic loci. A total of 99 RAPD markers were evaluated of which 67 (67.67 percent) were polymorphic. AMOVA showed that 38.5 percent and 61.5 percent of the genetic variation was distributed among and within populations, respectively. The fixation index (F ST) of the genotypes was 0.385. The mean genetic variability estimated within populations ranged from 15.58 (IAPAR 59) to 8.27 (Catuaí Vermelho IAC 81). A distinct level of genetic variability was revealed for each of the coffee progenies and varieties studied. The methodology used in this investigation was useful to determine the genetic variability within and among C. arabica L. populations providing significant information for coffee breeding