Paulo Maurício Ruas

Genetic relationship in Coffea species and parentage determination of interspecific hybrids using ISSR (Inter- Simple Sequence Repeat) markers

Inter-simple sequence repeat (ISSR) markers were used to evaluate genetic divergence among eight Coffea species and to identify the parentage of six interspecific hybrids. A total of 14 primers which contained different simple sequence repeats (SSR) were used as single primers or combined in pairs and tested for PCR amplifications. Two hundred and thirty highly reproducible fragments were amplified, which were then used to estimate the genetic similarity and to cluster the Coffea species and hybrids. High levels of interspecific genetic variation were revealed. The dinucleotide motif (GA)9T combined with other di- tri- and tetra-nucleotides produced a greater number of DNA fragments, mostly polymorphics, suggesting a high frequency of the poly GA microsatellite motifs in the Coffea genomes. The genetic similarity ranged from 0.25 between C. racemosa and C. liberica var. dewevrei to 0.86 between C. arabica var. arabica and Hybrid N. 2. The C. arabica species shared most of its markers with five of the six hybrids suggesting that it is the most likely candidate as one of the progenitors of those hybrids. These results revealed that ISSR markers could be efficiently used for genetic differentiation of the Coffea species and to identify the parentage of Coffea interspecific hybrids.

Genetic diversity among maize (Zea mays L.) landraces assessed by RAPD markers

The genetic relationships among 81 maize accessions consisting 79 landraces and two improved varieties, maintained by farmers in southern Brazil were investigated using Random Amplified Polymorphic DNA (RAPD). Thirty-two highly informative primers amplified 255 markers of which 184 (72.2%) were polymorphics. Based on the RAPD markers, a dendrogram was constructed using the UPGMA method. The range of genetic similarity was from 0.78 to 0.91. The molecular data grouped the accessions into two main clusters, which were correlated according to kernel colors. Small clusters were seen associated to characteristics, such as kernel morphology. The analysis of the molecular data revealed that maize management adopted by small-scale farmers has contributed to the maintenance of genetic variability and since field isolation is a regular practice, variety identities have been preserved. These results will be useful to establish and maintain a germplasm collection of landrace maize and may guide us in designing strategies that maximize the utility of maize genetic resources.

Characterization of diploid, tetraploid and hexaploid Helianthus species by chromosome banding and FISH with 45S rDNA probe

Comparative karyotype analyses of five diploid, two tetraploid, and three hexaploid species of Helianthuswere performed using Feulgen staining, Giemsa C and CMA3 (C-CMA) staining, and FISH with 45S rDNA probe. The karyotypes are composed by a basic number of x = 17 with a predominance of meta- and submetacentric chromosome types. A polyploid series is associated with the basic number. Giemsa C- and C-CMA banding revealed terminal or interstitial heterochromatin according to the species, suggesting the existence of a mechanism that may be acting in the dispersion of heterochromatic segments in Helianthus. The nucleolar organizer regions were located at terminal chromosome positions by FISH with 45S rDNA probe. Diploid species presented four, six, and eight rDNA sites, tetraploid species showed eight sites and hexaploid species presented 12 rDNA sites. Karyomorphological differences include variation in number, size and chromosome morphology, suggesting that rearrangements involving small heterochromatic and rDNA segments played a major role in karyotype evolution.

Uso de marcadores moleculares na análise da variabilidade genética em acerola (Malpighia emarginata D.C.)

Acerola (Malpighia emarginata) is a tropical fruit native from Central America and north of South America. It has shown an increasing economic and social importance due to its high vitamin C (ascorbic acid) content. Vegetative propagation is the preferable method used to establish acerola plantations. However, propagation by seeds has also been used allowing the identification and selection of genotypes that carry characteristics of agronomic interest. Twenty-four acerola accesses, of the Active Germplasm Bank of the Universidade Estadual de Londrina, were analyzed using molecular markers obtained with random amplified polymorphic DNA (RAPD) and simple sequence repeated (SSRs) primers. A total of 164 and 73 markers were obtained with RAPD and SSR primers, respectively. Polymorfic markers were scored as present or absent and analyzed using the UPGMA cluster analysis. The results presented reveal high levels of polymorphism in the studied collection. Comparative analysis of the phenograms, generated with both RAPD and SSR primers, revealed that while some accesses clustered in different groups other accesses presented the same association. However, there was large RAPD variation among the accesses. The associations observed with molecular markers were, for many accesses, the same of those determined on the basis of morphological characters.

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 CMA 3 , 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.

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

Abstract TheRAPDtechniqueassociatedwithrestrictiondigestionofgenomicDNAwasusedtoassessthegeneticvariabilitywithin and among nine populations of Coffea arabica, including six progenies belonging to the Sarchimorgermplasm, the progeny PR 77054-40-10 (Catuai Vermelho IAC 81 x Icatu), and two commercial cultivars (IAPAR59andCatuaiVermelhoIAC-81).Thesepopulationswereevaluatedusinganalysisofmolecularvariance(AMOVA),genetic similarity among progenies, and percentage of polymorphic loci. A total of 99 RAPD markers were evaluatedof which 67 (67.67%) were polymorphic. AMOVA showed that 38.5% and 61.5% of the genetic variation wasdistributed among and within populations, respectively. The fixation index (F ST ) of the genotypes was 0.385. Themean genetic variability estimated within populations ranged from 15.58 (IAPAR 59) to 8.27 (Catuai Vermelho IAC81). A distinct level of genetic variability was revealed for each of the coffee progenies and varieties studied. Themethodology used in this investigation was useful to determine the genetic variability within and among C. arabicaL.populations providing significant information for coffee breeding.

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

RAPD was used to access the genetic variability in Aspisdosperma polyneuron, a long-lived, late-reproducing tropical tree, and highly important for the Atlantic Forest. RAPD profiles from adults (pre-fragmentation, >300 years old) and seedlings (post-fragmentation, <<50 years old) were analyzed. Results showed a decrease of genetic polymorphism of post-fragmentation cohorts in small fragments and higher genetic diversity within population. The genetic diversity distribution suggested the establishment of fragments as protected reserves, and the transference of seedlings among fragments for conservation of A. polyneuron.

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%) were polymorphic. The amplified products were used to estimate the genetic variability using Dices 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.

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

ABSTRACT 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% and for the SSR from 11 to 82%. 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. Key words: Corn, distance genetic, microsatellites, molecular markers, polymorphism, RAPD

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

The genetic variability of 40 accessions of_C. arabica was evaluated using a combination of the random amplified polymorphic DNA (RAPD) technique and restriction digestion of genomic DNA. The genetic variability and the relatedness among all accessions were initially evaluated using 195 RAPD primers which revealed a very low level of genetic variation. To improve the efficiency in the detection of polymorphism, the genomic DNA of all accessions were submitted to digestion with restriction endonucleases prior to PCR amplification. A total of 24 primers combined with restriction digestion of DNA rendered 318 bands, of which 266 (83.65%) were polymorphic. The associations among genotypes were estimated using UPGMA-clustering analysis. The accessions were properly clustered according to pedigree and agronomic features. The ability to distinguish among coffee accessions was greater for RAPD plus restriction digestion than for RAPD alone, providing evidences that the combination of the techniques was very efficient for the estimation of genetic relationship among_C. arabica genotypes.