Development and characterization of microsatellite loci for Campomanesia xanthocarpa (Myrtaceae) and cross amplification in related species

Campomanesia xanthocarpa is a native tree, of common occurrence in almost all Brazilian Forest formations, which has its fruits and timber with high commercial value. Using an enriched genomic library we isolated and characterized microsatellite loci for C. xanthocarpa (Myrtaceae), in order to estimate genetic diversity parameters for this and related species. Twenty-eight microsatellite loci were identified and ten of them successfully amplified and showed polymorphism in a sample of 96 individuals, from four natural populations. The number of alleles per locus ranged from two to eight, and the observed and expected heterozygosities varied from 0.042 to 1.000 and from 0.294 to 0.855, respectively. These markers were tested and validated in two related species (C. eugenioides and C. guazumifolia). The microsatellite markers will be used in further studies of population genetics of C. xanthocarpa, in order to understand the genetic variability and to define the strategies needed for the conservation of the species.

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.

Progênies de Coffea arabica cv. IPR 100 com resistência ao nematóide Meloidogyne incognita raça 1 / Progenies of Coffea arabica cv. IPR 100 with resistance to nematode Meloidogyne incognita race 1

Objetivou-se, neste trabalho, identificar progênies de Coffea arabica cv. IPR 100 com resistência ao nematóide Meloidogyne incognita raça 1. O experimento foi conduzido em casa de vegetação (Londrina, PR, Brasil) no delineamento em blocos ao acaso com 3 repetições e parcelas com 15 plantas. Foram avaliadas seis progênies da cultivar IPR 100 e a cultivar Mundo Novo IAC 376-4 foi utilizada como padrão suscetível. Foram inoculados 500 ovos por planta, totalizando 7500 ovos por parcela de 150 cm². Foi avaliado o número de galhas e massas de ovos presentes nas raízes. As seis progênies da cultivar IPR 100 foram mais resistentes ao M.incognita raça 1 do que o padrão suscetível.

The objective of this study was to identify progenies of Coffea arabica cv. IPR 100 with resistance to nematode Meloidogyne incognita race 1. The experiment was performed in greenhouse (Londrina, PR, Brazil) with an experimental design of randomized blocks with 3 replications and 15 plants per plot. Six progenies of cultivar IPR 100 were evaluated, and Mundo Novo IAC 376-4 cultivar was used as susceptible control. Five hundred eggs per plant were inoculated, totalizing 7500 eggs per plot of 150 cm². The number of galls and egg masses in the roots were evaluated. All progenies of cultivar IPR 100 presented more resistance to M.incognita race 1 than the susceptible control.

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 control of orange hilum corona of carioca beans (Phaseolus vulgaris)

The purpose of this research was to elucidate the genetic control of orange corona color in carioca common beans (Phaseolus vulgaris). We made four crosses between carioca group cultivars that differed in respect to the presence or absence of an orange hilum corona color. The F2, F3, F1BC11, F1BC21, F2BC11 and F2BC21 phenotypic segregations were evaluated with a chi-square test which fitted with the hypothesis that one gene with a dominant allele is responsible for the orange corona color. All generations resulting from the four different crosses showed segregation patterns which agreed with the expected proportions. Our results show that the dominant G allele controls orange corona color in the carioca bean group.

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