Development of microsatellite markers for Myracrodruon urundeuva (F.F. & M.F. Allemão), a highly endangered species from tropical forest based on next-generation sequencing.

Myracrodruon urundeuva is a tree species of high economic importance due the strength and durability of its wood. Threatened of extinction in Brazil, it is present only in a few forest remnants, mostly in conservation units. Currently, there is little information on the genetic diversity of natural populations in Brazil and even less information about the genome of this species. Here, new species-specific microsatellite loci were developed based on next-generation sequencing (Illumina). More than 100,000 loci were identified in the run, with di- to hexanucleotides motifs. Of these, 20 loci were selected for validation in 30 individuals, with 15 successfully polymorphic loci detected. The number of alleles ranged among loci from 3 to 16, with an average of 7.73, expected (H e ) and observed (H o ) heterozygosity ranged from 0.246 to 0.902 and from 0.103 to 0.867, respectively. These results point out that these new set of markers has a great potential for use in population genetic studies for genetic conservation of the species.

Spatial genetic structure, genetic diversity and pollen dispersal in a harvested population of Astrocaryum aculeatum in the Brazilian Amazon.

BACKGROUND: Astrocaryum aculeatum is a palm tree species native to the tropical regions of South America, exploited commercially by local farmers for the pulp extracted from its fruits. The objective of this research was to compare the genetic diversity between adult plants and seedlings from open-pollinated seeds, quantify the pollen flow and dispersal, the spatial genetic structure, and the effective size of a population that has been continuously harvested for its fruits. The study was carried out in a natural population of A. aculeatum distributed over approximately 8 ha in the State of Amazonas (Brazil), separated by 400 m from the closest neighboring population. In total, 112 potential pollen donors, 12 mother plants and 120 offspring were mapped and genotyped. RESULTS: Genetic diversity was high for parents and the offspring. The fixation indexes for adults (F = -0.035) and offspring (F = -0.060) were negative and not significant. A significant spatial genetic structure was detected for the adult plants (up to the distance of 45 m) indicating short-distance seed dispersal. Paternity analysis detected 9.2 % of pollen immigration and the average distance of pollination within the population was 81 m. The average effective pollination neighborhood area between plants was 1.51 ha. CONCLUSIONS: Our results indicate that substantial introduction of new alleles has occurred in the population through pollen immigration, contributing to the maintenance of genetic diversity. Conservation efforts aimed at maintaining the gene pool of the current population or establishing new populations should utilize offspring from mother plants selected to be spaced by at least 50 m to prevent collecting seeds from relatives.

Diversity and spatial genetic structure of a natural population of Theobroma speciosum (Malvaceae) in the Brazilian Amazon.

The quantification of genetic diversity and intrapopulation spatial genetic structure (SGS) of tree species are important aspects for in and ex situ conservation practices. In this study we seek to understand the importance of conservation areas by quantifying the genetic diversity and the spatial genetic structure of a natural population of Theobroma speciosum. Within this population, 49 adults and 51 subadults were genotyped for five microsatellite loci. The results showed that adults and subadults have similar levels of genetic diversity and inbreeding (adults: A= 10.4, Ae = 10.3, F= 0.68, subadults: A= 10.6, Ae= 10.6, F= 0.57). Genetic diversity was spatially structured within the population, and the results suggest that near-neighbor trees up to a distance of 70 m are likely related. SGS is likely the result of short-distance seed dispersal, the short-distance range of pollinators, and infrequent breaches of the self-incompatible mating system. Considering the high demographic density of the species and size of the study area, as well as the high average number of alleles per locus and the presence of rare alleles, we believe that the study population is an excellent resource for in situ genetic conservation of T. speciosum. The study area is also a useful resource for collecting germplasm for ex situ conservation and seed collection, either for breeding programs used in the restoration of degraded areas or forest improvement.

Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from Theobroma seeds on midgut protease activity of Lepidopteran pest insects.

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

Natural Populations of Astrocaryum aculeatum Meyer in Amazonia: Genetic Diversity and Conservation.

Astrocaryum aculeatum, a palm tree incipiently domesticated from upland ecosystems in the Brazilian Amazon, is especially adapted to anthropized areas. The pulp of the fruit, obtained by extractivism, is consumed fresh by the Amazonian population. The objective of the study is to evaluate the diversity and genetic structure of the natural populations of A. aculeatum, exploited by extractive farmers in Amazonas, Brazil, seeking to suggest conservation and management strategies for this species. A total of 218 plants were sampled in 15 populations in 14 municipalities in the state of Amazonas, evaluated by 12 microsatellite loci. A total of 101 alleles were observed. The means of the observed heterozygosities (HO = 0.6390) were higher than expected (HE = 0.557), with high levels of heterozygotes in the populations. The fixation index in the loci and populations was negative. The FST (0.07) and AMOVA showed moderate population structure. Bayesian analysis indicated the grouping k = 4 as the most adequate. There is a high genetic diversity in populations, with a moderate genetic structure due to possible historical events, which could be related to the process of subpopulation formation, possibly presenting three historical moments: before and after the beginning of deforestation and today. The conservation and management policies of this species must be carried out at a watershed level.

Paternity analysis, pollen flow, and spatial genetic structure of a natural population of Euterpe precatoria in the Brazilian Amazon.

Euterpe precatoria, known as açaí do Amazonas, is a regionally important palm of the Amazon rainforest for the fruit production through extractive agriculture. Little information is available with regard to genetic diversity, gene flow, and spatial genetic structure (SGS) of açaí populations, which are essential for the use, management, and conservation of genetic resources of the species. This research aimed to assess the genetic diversity, inbreeding level, SGS, and gene flow in four ontogenetic stages of a natural E. precatoria population in the Brazilian Amazon, based on 18 microsatellite loci. The study was carried out in a natural population dispersed in an area of about 10 ha. Leaf tissues of 248 plants were mapped and sampled and classified into four ontogenetic stages: reproductive (59), immature (70), young (60), and seedling (59). Genetic diversity indices were high for all ontogenetic stages. The fixation index (F) for all ontogenetic stages was not significantly different from zero, indicating the absence of inbreeding. A significant SGS was found for all ontogenetic stages (68-110 m), indicating seed dispersal over short distances. Paternity analysis detected pollen immigration of 39.1%, a selfing rate of 4.2%, and a mean pollen dispersal distance within the population of 531 m. The results indicate substantial allele input in the population via pollen immigration, contributing to the maintenance of the genetic diversity of the population. However, within a population, the renewal with new progenies selected from seed plants spaced at least 110 m apart is important to avoid collecting seeds from related plants.

Inferring mating system parameters in forage peanut, Arachis pintoi, for Brazilian Amazon conditions

The search for alternatives to increase productivity and sustainability of livestock production in the Amazon region without increasing deforestation is challenging. Mixed pastures of grasses with forage peanut (Arachis pintoi) have shown positive economic impacts. However, gaps in the knowledge of the reproductive biology of A. pintoi have limited the development of new cultivars adapted to the environmental variations in the Brazilian Amazon. Pasture consortiums of Brachiaria humidicola with forage peanuts (cv. Mandobi) resulted in a 42% increase in weight gain productivity. New cultivars better adapted to the Amazon climate should bring even greater gains. We evaluated the mating system in twenty A. pintoi accessions, and approximately 40 offspring per accession genotyped with eight microsatellites (or markers). The parameters of genetic diversity and inbreeding, the outcrossing rate and coancestry were calculated. The observed heterozygosity was significantly higher and the fixation index was significantly lower in adults compared with the offspring. The crossing rate was variable among genotypes (2 to 80%), and the mean outcrossing rate was 36%. These results indicate that pollinator presence in pastures can influence gene flow in A. pintoi more than expected. Arachis pintoi presented a mixed mating system with a predominance of selfing, and families presented inbreeding and different levels of relatedness. New strategies of genotype conservation are needed to avoid pollinator-mediated crossing between accessions. (AU)

Microsatellite markers for Cariniana legalis (Lecythidaceae) and their transferability to C. estrellensis.

PREMISE OF THE STUDY: Microsatellite primers were developed in the neotropical tree species Cariniana legalis (Lecythidaceae) to investigate its genetic diversity, mating system, and gene flow. • METHODS AND RESULTS: We identified 96 clones containing 82 repeat motifs from a genomic library enriched for (CT)8 and (GT)8 motifs. Primer pairs were developed for 13 microsatellite loci and validated in 51 C. legalis specimens and 26 C. estrellensis specimens. Eleven loci were polymorphic, revealing a maximum of two to 15 alleles per locus in C. legalis and three to 12 in C. estrellensis. For C. legalis, the observed (H o) and expected (H e) heterozygosities ranged from 0 to 0.99 and from 0.07 to 0.90, respectively. For C. estrellensis, H o and H e ranged from 0 to 0.96 and from 0.14 to 0.91, respectively. • CONCLUSIONS: The primers identified polymorphic loci that are suitable to study genetic diversity and structure, mating system, and gene flow in C. legalis and the related species C. estrellensis.

Inheritance and linkage relationships of allozyme variants of ilex paraguariensis St. Hil

Eighteen enzyme systems were examined in Ilex paraguariensis St. Hil. using starch gel electrophoresis. Seven out of 12 active isozyme systems revealed one or more polymorphic loci (PGI, GOT, MR, G-6PDH, MDH, NDH, and 6-PGDH). However, the segregation and linkage analyses were performed only for PGI, GOT, G-6PDH and 6-PGDH systems. Gene segregation at these loci was regular, except for a few trees that showed segregation distortion. Weak linkage disequilibrium between loci was detected, but it was not enough to influence the multilocus estimate.

Dezoito sistemas enzimáticos foram analisados em Ilex paraguariensis, utilizando eletroforese em gel de amido. Sete dos 12 sistemas que apresentaram atividade enzimática revelaram um ou mais locos polimórficos (PGI, GOT, MR, G-6PDH, MDH, NDH e 6-PGDH). Entretanto, as análises de segregação e desequilíbrio de ligação foram realizadas somente nos sistemas PGI, GOT, G-6PDH e 6-PGDH. A segregação destes locos foi regular, exceto para algumas árvores, que apresentaram distorções de segregação. Foram detectados indícios de desequilíbrio de ligação entre alguns locos, mas que não devem influenciar as estimativas multilocos.