Claudete de Fátima 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.

AFLP Phylogeny of South American Species of Hypochaeris (Asteraceae, Lactuceae)

Abstract Hypochaeris is thought to have arrived in South America by dispersal over the Atlantic Ocean from NW Africa during the Pliocene or Pleistocene. We used amplified fragment length polymorphism (AFLP) to unravel specific limits and relationships in the South American group of Hypochaeris (c. 45 species). The Moroccan endemic Hypochaeris angustifolia, which is sister to the entire South American group, was used as outgroup. Our AFLP analysis comprises 415 individuals from 32 South American species and is based on six primer combinations with 670 fragments scored. It provides important information for the delimitation of species and detection of closely related species pairs or groups. Most species are monophyletic and supported with > 90% bootstrap proportion. Hybridization is suggested between Hypochaeris chillensis and H. microcephala in Brazil. The internal nodes (or “backbone”) of the tree are not highly supported, but six major phylogenetic groups (also showing similarity in distribution and growth form) center around Hypochaeris apargioides, H. chondrilloides, H. microcephala, H. pampasica, H. sessiliflora, and H. tenuifolia. These results suggest that rapid migration into different geographical regions played an important role in the initial diversification of Hypochaeris in South America.

Molecular phylogenetics reveals Leontodon (Asteraceae, Lactuceae) to be diphyletic

The plastid matK gene, trnL/F spacer, and nuclear rDNA ITS were sequenced for 36 species of Leontodon and 29 taxa of related genera of tribe Lactuceae. Phylogenetic relationships inferred from the independent and combined data are largely congruent and reveal that Leontodon sensu lato (s.l.) as presently defined is diphyletic: L. subgenus Leontodon forms a clade with Helminthotheca, Picris and Hypochaeris as sister genera, whereas L. subgenus Oporinia appears as a separate clade with strong bootstrap support and is thus better treated as a separate genus. Previous sectional classifications of Leontodon s.l. are considered in the light of DNA and additional morphological and karyological data. Support is presented for a core group of Hypochaeridinae sensu stricto (s.s.) with the two clades of Leontodon s.l., Helminthotheca, Picris, and Hypochaeris, whereas Urospermum, Hyoseris, Aposeris, and Rhagadiolus appear to be positioned more distantly.

Phylogeography of the invasive weed Hypochaeris radicata (Asteraceae): from Moroccan origin to worldwide introduced populations

In an attempt to delineate the area of origin and migratory expansion of the highly successful invasive weedy species Hypochaeris radicata, we analysed amplified fragment length polymorphisms from samples taken from 44 populations. Population sampling focused on the central and western Mediterranean area, but also included sites from Northern Spain, Western and Central Europe, Southeast Asia and South America. The six primer combinations applied to 213 individuals generated a total of 517 fragments of which 513 (99.2%) were polymorphic. The neighbour‐joining tree presented five clusters and these divisions were supported by the results of Bayesian analyses: plants in the Moroccan, Betic Sierras (Southern Spain), and central Mediterranean clusters are all heterocarpic. The north and central Spanish, southwestern Sierra Morena, and Central European, Asian and South American cluster contain both heterocarpic (southwestern Sierra Morena) and homocarpic populations (all other populations). The Doñana cluster includes two homocarpic populations. Analyses of fragment parameters indicate that the oldest populations of H. radicata are located in Morocco and that the species expanded from this area in the Late Quaternary via at least three migratory routes, the earliest of which seems to have been to the southwestern Iberian Peninsula, with subsequent colonizations to the central Mediterranean area and the Betic Sierras. Homocarpic populations originated in the southwestern Iberian Peninsula and subsequently spread across north and central Spain, Central Europe and worldwide, where they became a highly successful weed.

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.