Eduardo Augusto 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 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.

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.

Flooding tolerance and genetic diversity in populations of Luehea divaricata

We investigated some aspects of flooding tolerance in two riparian populations (exposed and no exposed to flooding) of Luehea divaricata C. Martius. Plants derived from seeds collected in each population were submitted to flooding (30 and 60 d), submergence and re-aeration treatments. Plants exposed to flooding showed development of aerenchyma, hypertrophic lenticels and new adventitious roots. Interestingly, whereas the plants originated from population naturally exposed to flooding developed some of these alterations more markedly, they could not survive when totally submerged. The random amplified polymorphic DNA (RAPD) markers, showed a significant difference between populations, suggesting that seasonal flooding on riparian populations of L. divaricata has been selecting individuals who are more adapted to survive in these conditions.

AFLP marker analysis revealing genetic structure of the tree Parapiptadenia rigida (Benth.) Brenan (Leguminosae-Mimosoideae) in the southern Brazilian Tropical Rainforest

Parapiptadenia rigida is a tropical early secondary succession tree characteristic of the Tropical Atlantic Rainforest. This species is of great ecological importance in the recovery of degraded areas. In this study we investigated the variability and population genetic structure of eight populations of P. rigida. Five AFLP primer combinations were used in a sample of 159 individuals representing these eight populations, rendering a total of 126 polymorphic fragments. The averages of percentage of polymorphic loci, gene diversity, and Shannon index were 60.45%, 0.217, and 0.322, respectively. A significant correlation between the population genetic variability and the population sizes was observed. The genetic variability within populations (72.20%) was higher than between these (22.80%). No perfect correlation was observed between geographic and genetic distances, which might be explained by differences in deforestation intensities that occurred in these areas. A dendrogram constructed by the UPGMA method revealed the formation of two clusters, these also confirmed by Bayesian analysis for the number of K cluster. These results show that it is necessary to develop urgent management strategies for the conservation of certain populations of P. rigida, while other populations still preserve reasonably high levels of genetic variability.

Genetic structure of the Atlantic Rainforest tree species Luehea divaricata (Malvaceae)

The Atlantic Rain Forest is one of the most important Brazilian biomes and a hotspot for biodiversity that is characterized by its high level of endemism, where new species are still being described. Luehea divaricata (Malvaceae) is commonly found in riparian forests areas of the Atlantic forest. Because of the importance of this species in reforestation programs, we used nine pairs of microsatellite loci to study the genetic variability of this species along its distribution area and verify if fragmentation is compromising the survival of these populations. A total of 50 alleles were obtained with an average observed and expected heterozygosity of 0.53 and 0.67, respectively. Seven of the nine populations studied showed a heterozygosity deficit. Most of the genetic diversity was found within populations; while the level of genetic differentiation was moderated (6.84) between populations. Different levels of gene flow between the populations were detected. Positive and significant values of Fis were found for seven populations. The signal test for excess of heterozygosity indicated that a recent genetic bottleneck occurred in the fragmented populations. The dendrogram constructed by the UPGMA method revealed the formation of seven clusters, which was confirmed by the Bayesian analysis for number of K clusters. The presence of several pairs of loci in linkage disequilibrium confirms that these populations experienced a loss of genetic diversity caused by genetic drift. The results showed that it is necessary to develop management strategies for the conservation of these populations of L. divaricata as the viability of the next generations are severely compromised.

Morfoanatomia de órgãos vegetativos de plantas juvenis de Aegiphila sellowiana Cham. (Lamiaceae) submetidas ao alagamento do substrato

In order to uncover the morpho-anatomical responses of tree species to hypoxia, we studied the effects of flooding on vegetative organs of Aegiphila sellowiana. Young individuals were kept in drained and flooded soil for 50 and 80 days. Under flooding, there was a mortality rate of 40%. The surviving plants produced peridermic fissures, superficial roots and hypertrophied lenticels. Secondary roots of flooded plants presented greater diameter, thicker cortex, greater diameter of the cortical cells, larger intercellular spaces, and thinner vessel elements and vascular tissues. In the main roots under secondary growth and hypocotyls, cork and phelloderm became thicker under flooding. Leaves of flooded plants presented thicker midrib, larger mesophyll, higher number of trichomes and smaller stomata. Considering that part of the plants subject to flooding have died, it is possible to suggest that A. sellowiana is intolerant of soil inundation. However, the surviving plants showed morpho-anatomical modifications that could have conferred some tolerance to them. Thus, the differential tolerance observed among individuals of A. sellowiana indicates a variability of genotypes in the samples used in this study.

Genetic Structure and Diversity of Brazilian Tree Species from Forest Fragments and Riparian Woods

Understanding the genetic structure of populations that occur in forest remnants is fundamentally necessary to establish efficient strategies for the re-composition, management, and conservation programs. For such, it is necessary not only to understand the genetic diversity of a species, but also, how this diversity is distributed within and between forest populations. Notably, a considerable part, if not the majority, of Brazilian Atlantic Forest fragments are linked to rivers or streams, once the policy applied in Brazil regarding conservation in agricultural areas favours the maintenance of legal reserves in proximity of water sources. The vegetation of river margins are subjected to flooding, a strong limiting factor which can lead to local adaptation. These ecological and landscape characteristics may have important outcomes to the genetic diversity of tree populations.

Isolation and characterization of eleven polymorphic microsatellite loci in Aegiphila sellowiana and their transferability

We isolated and characterized eleven polymorphic microsatellite loci for Aegiphila sellowiana an outcrossing pioneer tree species that is frequently used in reforestation programs of tropical riparian forests in Brazil. A total of 38 alleles were detected across a sample of 45 individuals of A. sellowiana, with an average number of 3.45 alleles per locus. The average polymorphic information content (PIC) was 0.430 and the observed (HO) and expected (HE) heterozygosity values varied from 0.156 to 1.000 and 0.145 to 0.730, respectively. Eight loci exhibited significant deviation from Hardy-Weinberg equilibrium (P ≤ 0.001) and 32 pair combinations of loci showed significant linkage disequilibrium (P ≤ 0.001). All 11 primers were tested for cross amplification in 12 species belonging to the family Lamiaceae and 5 species belonging to the related family Verbenaceae. The sequence and diversity information obtained using these microsatellites and their cross-transferability to other species of Lamiaceae as well as Verbenaceae will increase our understanding of genetic structures and species relationships within Aegiphyla and other genera of these families.

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, Parana, 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% of the genetic variation was among the population. Pairwise FST 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 FST and indicating that for conservation purposes all populations should be maintained.