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- Genetic diversity and hybridization in the two species Inga ingoides and Inga edulis: potential applications for agroforestry in the Peruvian AmazonPublication . Rollo, Alexandr; Lojka, Bohdan; Honys, David; Mandák, Bohumil; Wong, Julio A. Chia; Santos, Carmen; Costa, Rita; Quintela-Sabarís, Celestino; Ribeiro, M.M.A.Key message: Slash and burn practices affect tropical forests. Our results showed strong introgression between Inga ingoides and Inga edulis in the species contact area Interspecific hybridization could be sought to improve yield or tolerance to flooding and further increase the economic potential of the poorly drained Amazonian soils and minimize deforestation. Context: Inga species are important components of tropical American forests, as well as a local food source. Little is known about the genetic structure of these species; in particular the amount of introgression among species remains unknown. Aims: We assessed the degree of genetic divergence and introgression among populations of I. ingoides (Rich.) Willd. and I. edulis Mart. (Fabaceae) from three Peruvian Amazon tributary rivers. Methods: Using microsatellite markers we determined the genetic structure of populations using an analysis ofmolecular variance and a Bayesian analysis of population structure in areas affected by seasonal river fluctuations and in ‘terra firme’ forests. Results: Overall genetic differentiation was weak. The degree of genetic variation was similar in the two species. A putatively strong introgression was detected between the two species and an intense gene flow was identified among populations. This indicates that an intense gene flow had happened in the past, leading also to a small differentiation among populations within species. Conclusion: Selection of natural hybrids or artificial hybridization between I. edulis and I. ingoides could be applied to improve legume size and yield in the later species, while maintaining tolerance to flooding. Improved I. ingoides could be used in multipurpose agroforestry on open areas along the rivers, instead of using the usual slash and burn practice to create inland open areas.
- Genetic structure and pod morphology of Inga edulis cultivated vs. wild populations from the Peruvian AmazonPublication . Rollo, Alexandr; Ribeiro, M.M.A.; Costa, Rita; Santos, Carmen; Clavo P., Zoyla M.; Mandak, Bohumil; Kalousová, Marie; Vebrová, Hana; Chuqulin, Edilberto; Torres, Sergio G.; Aguilar, Roel M.V.; Hlavsa, Tomáš; Lojka, BohdanResearch Highlights: This study assesses the genetic diversity and structure of the ice-cream-bean (Inga edulis Mart.) (Fabaceae), in wild and cultivated populations from the Peruvian Amazon. This research also highlights the importance of protecting the biodiversity of the forest in the Peruvian Amazon, to preserve the genetic resources of species and allow further genetic improvement. Background and Objectives: Ice-cream-bean is one of the most commonly used species in the Amazon region for its fruits and for shading protection of other species (e.g.: cocoa and coffee plantations). Comprehensive studies about the impact of domestication on this species’ genetic diversity are needed, to find the best conservation and improvement strategies. Materials and Methods: In the current study, the genetic structure and diversity were assessed by genotyping 259 trees, sampled in five wild and 22 cultivated I. edulis populations in the Peruvian Amazon, with microsatellite markers. Pod length was measured in wild and cultivated trees. Results: The average pod length in cultivated trees was significantly higher than that in wild trees. The expected genetic diversity and the average number of alleles was higher in the wild compared to the cultivated populations, thus, a loss of genetic diversity was confirmed in the cultivated populations. The cultivated trees in the Loreto region had the highest pod length and lowest allelic richness, nevertheless the wild populations’ genetic structure was not clearly differentiated (significantly different) from that of the cultivated populations. Conclusions: A loss of genetic diversity was confirmed in the cultivated populations. The species could have been simultaneously domesticated in multiple locations, usually from local origin. The original I. edulis Amazonian germplasm should be maintained. Cultivated populations’ new germplasm influx from wild populations should be undertaken to increase genetic diversity.
- Genetic diversity and divergence at the Arbutus unedo L. (Ericaceae) westernmost distribution limitPublication . Ribeiro, M.M.A.; Piotti, A.; Ricardo, A.; Gaspar, Daniel; Costa, Rita; Parducci, L.; Vendramin, Giovanni GiuseppeMediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species' distribution range in the region. We applied Bayesian analysis and identified four clusters (north, centre, south, and a single-population cluster). Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species' genetic structure.
- DNA markers to study genetic diversity and fingerprinting in Arbutus unedo L. (strawberry tree)Publication . Gomes, F.; Costa, Rita; Ribeiro, M.M.A.; Figueiredo, Elisa; Canhoto, JorgeStrawberry tree (Ericaceae) is a species of Mediterranean climates growing in several countries of the Mediterranean basin as well as in Portugal and Ireland. The edible fruits have been used in different ways yet the production of a popular spirit is still the main application. The species is also interesting from an ecological perspective since it is drought tolerant and fire resistant. In spite of its large distribution in Portugal little is known about the genetic diversity of the species and no efforts have been carried out to select the most interesting trees. Material from selected trees (based on fruit production) in a total of 27 was collected all around the country covering the distribution area of the species. In order to find out DNA markers to characterise genetic diversity and to fingerprint Arbutus unedo, random amplified polymorphic DNA (RAPD) and microsatellite markers (SSR) were used. The set of 20 RAPD primers used generated 124 bands, from which 71 (57.3%) were polymorphic and the expected heterozygosity was 0.27±0.014. The cluster analysis (UPGMA) based on the Jaccard similarity index showed a similarity of 71% among the trees of different origins. Only four genotypes could be grouped according to their geographical distribution. Eleven SSR primers designed for Vaccinium (an Ericaceae as A. unedo) were selected according to their polymorphism, number of alleles per locus, cross-amplification, allele scoring quality and the repeat motif, and were tested in A. unedo. Six loci showed to be polymorphic, with 71% mean expected diversity, 10.5 mean number of alleles, and 67% of average polymorphic information content. A mean homozygote excess was found (11%) although not significant. Estimation of null allele frequency was about 9% on average. The linkage disequilibrium test showed that all the loci were independent. The UPGMA clustering based on the Lynch and Ritland coefficient showed that only two genotypes shared about 50% of the alleles, but without a consistent geographic pattern, similarly to the results obtained by RAPD analysis. The Mantel test confirmed that there is no correlation between the genetic and the geographical distance matrices, for both makers. Reduced gene flow, due to fragmentary distribution, together with the type of mating system of this species (pollination by small insects), are plausible explanations for these results. As far as we know our work is the first detailed study to evaluate A. unedo diversity by molecular markers opening new perspectives for fingerprinting analysis and for conservation and improvement purposes.
- Procurando loci associados a variáveis do solo através de um varrimento molecular exploratório do genoma no pseudometalófito Cistus ladanifer L.Publication . Quintela-Sabarís, Celestino; Ribeiro, M.M.A.; Costa, Rita; Fraga, M. IsabelO uso do varrimento molecular do genoma juntamente com dados ambientais dá-nos a possibilidade de identificar marcadores moleculares com relevância ambiental. Neste trabalho foram analisadas 33 populações do pseudometalófito Mediterrâneo Cistus ladanifer L. (Cistaceae) com marcadores moleculares AFLP. Aplicámos Equações de Estimação Generalizadas (GEE) para estudar a correlação entre a distribuição de alelos AFLP e as diferentes características dos solos (pH, razão Ca:Mg e teores totais dos elementos vestigiais Mn, Ni, Pb, Sb e Zn). A análise GEE incluiu informação prévia das linhagens cloroplastidiais para eliminar o efeito da filogeografia. Eliminámos, também, os falsos positivos através do teste de Mann-Whitney e da observação da distribuição geográfica dos alelos. Os resultados mostraram que os teores totais de Mn têm influência na distribuição de alelos de C. ladanifer e que esse efeito foi o mais elevado relativamente a todas as outras variáveis do solo. Pelo contrário, a razão Ca:Mg parece não ter nenhum efeito selectivo em C. ladanifer, o que é confirmado pelo conhecimento relativo às suas necessidades em cálcio. Finalmente, detectámos um alelo possivelmente relacionado com a tolerância de C. ladanifer a solos com elevadas concentrações de Mn. Esta informação pode ser usada na selecção de indivíduos tolerantes a elevadas concentrações de metais tóxicos, Mn em particular, para uso na fitorremediação de solos contaminados.
- Application of biophysical factors and molecular markers to explain spatial genetic structure in strawberry tree using GIS toolsPublication . Ribeiro, M.M.A.; Quinta-Nova, L.C.; Roque, Natália; Ricardo, Alexandra; Gaspar, Daniel; Costa, RitaThe strawberry tree (Arbutus unedo L.) is a native species, water stress and low fertility soils tolerant, actively resistant to wildfires and widely distributed in Portugal. The fruit is used in the spirit production, the main source of income. Red fruits, with antioxidant potential, represent, also, a new market opportunity. The geographical isolation and extinction-recolonization dynamics are two factors causing strong genetic structure in metapopulations. We investigated how history, geography, and geoclimatic factors have affected population genetic structure, local adaptation, and, ultimately, its phylogeography. We examined patterns and levels of genetic diversity with nuclear microsatellites and cpDNA haplotypes in populations from across the species range. Under the project ARBUTUS (PTDC/AGR-FOR/3746/2012, Arbutus unedo plants and products quality improvement for the agro-forestry sector) 30 trees were selected, georeferenced, and leaves sampled, in 15 natural populations distributed throughout the country. With GIS tools, the stands were ecologically characterized, at a local scale, using lithology, topography, soil type, vegetation and wildfires records. This data was further used to distinguish Local Landscape Units (LLU) associated with each population. The populations were further clustered using large scale biogeographic and vegetation successions information and, this a priori hierarchy, together with the genetic structure information, was used to explain the species phytogeography. We aimed at finding the historical population demographic scenarios to explain the current patterns of genetic structure and diversity unfold for the species. The obtained information will be used in the species improvement, management and design of conservation programs.
- Genetic variability in wild and domestic populations of Inga edulis Mart. (Fabaceae) in Peruvian AmazonPublication . Rollo, Alexandr; Ribeiro, M.M.A.; Lojka, Bohdan; Honys, David; Sánchez Diáz, Henri Dante; Chia Wong, Julio Alberto; Vebrová, Hana; Costa, RitaHuman activity in the Peruvian Amazon causes native vegetation fragmentation into smaller units resulting on the increase of agricultural systems. Understanding the level, the structure and the origin of morphologic within and among populations variation is essential for planning better management strategies aimed at sustainable use and conservation of Inga edulis Mart. species. We evaluated the genetic variability in wild and domestic population to unfold cultivation changes over the species genetic resources. We have studied 400 adult trees: 200 cultivated on arable land and 200 wild growing in untouched lowland rain forest. The individuals were randomly selected. Sampling sites were selected and defined on the basis of the geographical coordinates: longitude, latitude and altitude. Phenotypic variation was monitored using the proposed descriptor of qualitative and quantitative features (e.g., weight of hundred seeds). For each individual a voucher specimen was kept. The total genomic DNA was extracted from young leaves, conserved in silica gel, with INVITEK, Invisorb ®Spin Plant Mini Kit. Samples were then genotyped with five microsatellite (SSR) loci. One locus (Pel5) was cross-transferred, developed previously for Pithecellobium elegans. The remaining four loci (Inga03, 05, 08, 33) were previously developed for the species. Polymerase chain reaction (PCR) was made using a Biometra® T1 Thermocycler using the following profile: 95 °C for 2 min; 95°C for 15 s, 55/59 °C for 30 s, 72 °C for 30 s, 30 cycles; 72 °C for 15 min. The PCR products were fluorescently labelled. The visualization of fragments was carried out according to standard protocols on genetic analyser, ABI PRISM® 310 (Applied Biosystems), using ABI GENESCAN and GENOTYPER software. The phenotypic and genotypic results of wild versus domestic populations are under evaluation to verify if cultivation is altering the allelic variation considering that morphology is considerably changed.