Jaqueline Bueno de Campos

Development of a novel set of microsatellite markers for Lippia alba (Verbenaceae)

Microsatellite primers were developed and optimized for Lippia alba to characterize the L. alba germplasm bank of Universidade de São Paulo. A genomic library enabled the design of 9 microsatellite primers. Six of the 9 primers yielded polymorphic products, which defined 2 groups in the bank. The data provide support to characterize germplasm banks, genetic breeding programs for L. alba, and other genetic diversity studies and classifications of species in the genus Lippia.

Molecular and chemical characterization of vetiver, Chrysopogon zizanioides (L.) Roberty, germplasm

Due to the economic interests in vetiver, Chrysopogon zizanioides (L.) Roberty, molecular and chemical studies are essential to generate information for its sustainable exploitation. The aim of this study was to undertake a molecular and chemical characterization of vetiver accessions of the active germplasm bank of the Universidade Federal de Sergipe. The molecular characteristics of the accessions were studied using amplified fragment length polymorphism markers, with a total of 14 primer combinations that generated 442 loci, allowing us to observe that these accessions have similar genomes. The vetiver accessions were divided into three distinct groups, where accession UFS-VET005 was the most differentiated and accession UFS-VET004 had the lowest essential oil content (0.70%). The content of the chemical constituents of the essential oils was observed to vary, with a predominance of khusimol, which ranged from 18.97 to 25.02%. It was possible to divide the vetiver accessions into two groups based on chemical composition, and these groups do not correlate with the molecular grouping. Therefore, it is necessary to perform molecular and chemical analyses to characterize vetiver accessions.

Genetic diversity of Lippia sidoides Cham. and L. gracilis Schauer germplasm.

The conservation of plants in germplasm banks ensures the characterization and availability of these resources for future generations. The present study used DNA markers to obtain genetic information about germplasm collections of Lippia sidoides and L. gracilis, which are maintained in an Active Germplasm Bank (AGB). Genetic variability of samples in the AGB was assessed using 12 combinations of amplified fragment length polymorphism (AFLP) primers (EcoRI/MseI). Twenty simple sequence repeat primers designed for L. alba were tested to determine their transferability in L. sidoides and L. gracilis. The AFLP markers generated 789 markers. The assessed loci exhibited a moderate Shannon diversity index (I = 0.42) in both species, suggesting that the conserved accessions possess an intermediate level of genetic diversity. Twelve microsatellite loci amplified satisfactorily, and nine loci were polymorphic in each species. A total of 23, 22, and 36 alleles, with an average of 2.5, 2.4, and 3.27 alleles per locus were identified for L. sidoides and L. gracilis accessions in the AGB, and Lippia sp sampled plants, respectively. Analyses of genetic structure permitted the identification of three different groups using both sets of markers, of which two were representative of L. sidoides. The information generated in this study may help to create, expand, and maintain collections of these species and may assist in genetic-breeding programs.

Cross-species amplification and polymorphism of microsatellite loci in Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) in Brazilian cropping systems

The Old World bollworm Helicoverpa armigera (Hübner) was recently discovered in Brazil. This species is closely related to the New World bollworm H. zea (Boddie), and mating between these species has already been reported under laboratory conditions. Here, we tested the cross-species amplification of 20 microsatellite (SSR) loci in field populations of H. armigera and H. zea collected from Brazilian cropping systems. Seven SSR loci were successfully amplified and polymorphic in both species except for the locus HaC14, which was monomorphic for H. zea. All SSR loci were in linkage equilibrium, and deviations from Hardy- Weinberg equilibrium were only observed for the locus HarSSR1 in the HaRS-2 population, where null alleles were present. A moderate level of polymorphism was detected in H. armigera and H. zea populations with a mean allele number of 4.14, and 2.24, respectively. Interestingly, most of the populations of the recent invader H. armigera showed higher genetic diversity and inbreeding coefficients than H. zea populations. The genetic identity of each species was recovered using a STRUCTURE analysis, where the populations formed two clusters (K = 2) according to their species. STRUCTURE also suggested the occurrence of potential hybrid offspring between H. armigera and H. zea individuals in natural conditions. These SSR loci will be valuable in characterizing population differentiation, invasion routes, adaptation, reproductive behavior, and intra- and interspecific gene flow in H. armigera and H. zea populations in Brazil, the USA, and other areas where these two pests occur.

Isolation and characterisation of microsatellite markers for Centrolobium tomentosum (Fabaceae), a neotropical tree species widely used for Atlantic Rainforest restoration

Abstract We isolated and characterised eight pairs of primers to amplify microsatellite regions for Centrolobium tomentosum, a neotropical tree species widely used for forest restoration, with important pharmacological potential. For the primer characterisation, we genotyped 48 individuals from two populations of C. tomentosum from natural remnants of Atlantic Rainforests. We detected 2–9 alleles per locus, observed and expected heterozygosities ranged from 0.08 to 0.72, and 0.08 to 0.83, respectively and we observed private alleles in six of the loci. No linkage disequilibrium was observed and all loci are in Hardy–Weinberg Equilibrium in at least one of the populations. This study presents a powerful tool for population genetic studies of this species.

Cross-species transferability of microsatellite markers in the genus Lippia.

The cross-species transferability of 20 microsatellite markers was tested in the genus Lippia. Eleven markers were polymorphic after screening 19 accessions of Lippia sidoides and Lippia gracilis maintained in the Active Germplasm Bank (AGB) from Universidade Federal de Sergipe. Additionally, 40 accessions of Lippia spp were collected in Sergipe to increase the germplasm bank. A total of 23, 22, and 36 alleles were identified, with an average of 2.3, 2.2, and 3.27 alleles per locus, respectively, for each group. The markers that were used were efficient tools to access genetic diversity in the germplasm bank and will be useful for further research aiming at the conservation and management of these important aromatic species.

Mating System and Effective Population Size of the Overexploited Neotropical Tree (Myroxylon peruiferum L.f.) and Their Impact on Seedling Production

The reproductive system of a tree species has substantial impact on genetic diversity and structure within and among natural populations. Such information, should be considered when planning tree planting for forest restoration. Here, we describe the mating system and genetic diversity of an overexploited Neotropical tree, Myroxylon peruiferum L.f. (Fabaceae) sampled from a forest remnant (10 seed trees and 200 seeds) and assess whether the effective population size of nursery-grown seedlings (148 seedlings) is sufficient to prevent inbreeding depression in reintroduced populations. Genetic analyses were performed based on 8 microsatellite loci. M. peruiferum presented a mixed mating system with evidence of biparental inbreeding (t^m-t^s = 0.118). We found low levels of genetic diversity for M. peruiferum species (allelic richness: 1.40 to 4.82; expected heterozygosity: 0.29 to 0.52). Based on Ne(v) within progeny, we suggest a sample size of 47 seed trees to achieve an effective population size of 100. The effective population sizes for the nursery-grown seedlings were much smaller Ne = 27.54-34.86) than that recommended for short term Ne ≥ 100) population conservation. Therefore, to obtain a reasonable genetic representation of native tree species and prevent problems associated with inbreeding depression, seedling production for restoration purposes may require a much larger sampling effort than is currently used, a problem that is further complicated by species with a mixed mating system. This study emphasizes the need to integrate species reproductive biology into seedling production programs and connect conservation genetics with ecological restoration.

Genetic diversity of Casearia sylvestris populations in remnants of the Atlantic Forest

Guaçatonga (Casearia sylvestris) is a native plant of the Atlantic Forest, with high medicinal potential and relevance for reforestation programs. The aim of this study was to characterize, with microsatellite markers, two populations of C. sylvestris from remaining areas of the Atlantic Forest in the State of São Paulo. High allelic variation was found in both populations (NA = 101 and 117; AR = 12.5 and 14.4), although with high endogamy coefficients (f = 0.640 and 0.363). Estimates of genetic structure suggested the presence of considerable genetic divergence between the populations (FST = 0.103); however, there was no spatial genetic structure within the populations. Genetic divergence may have occurred due to decreased gene flow between the fragmented populations as the result of deforestation. The results of this study demonstrate the importance of genetic diversity and its characterization in native plants within remaining forest areas for the management and restoration of such areas.

Development and Characterization of Microsatellite Markers for Piptadenia gonoacantha (Fabaceae)

Premise of the study: Microsatellite primers were designed for Piptadenia gonoacantha (Fabaceae) and characterized to estimate genetic diversity parameters. The species is a native tree from the Atlantic Forest biome commonly used in forest restoration; it has medicinal potential and the wood is economically useful. Methods and Results: Twenty-eight microsatellite loci were identified from an enriched genomic library. Fifteen loci resulted in successful amplifications and were characterized in a natural population of 94 individuals. Twelve loci were polymorphic, with allele numbers ranging from three to 15 per locus, and expected and observed heterozygosities ranging from 0.2142 to 0.8325 and 0.190 to 0.769, respectively. Conclusions: The developed markers will be used in further studies of population genetics of P. gonoacantha, aimed at conservation and management of the species in natural populations and in forest restoration projects.

Influence of historical land use and modern agricultural expansion on the spatial and ecological divergence of sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae) in Brazil

Human-mediated changes in landscapes can facilitate niche expansion and accelerate the adaptation of insect species. The interaction between the evolutionary history of the sugarcane borer, Diatraea saccharalis Fabricius, and historical and modern agricultural activity in Brazil shaped its spatial genetic structure, facilitating ecological divergence and incipient host shifting. Based on microsatellite data, STRUCTURE analyses identified two (K = 2) and three (K = 3) significant genetic clusters that corresponded to: (a) a strong signal of spatial genetic structure and, (b) a cryptic signal of host differentiation. We inferred that K = 2 reflects the footprint of agricultural activity, such as expansion of crop production (sugarcane and maize), unintentional dispersion of pests, and management practices. In contrast, K = 3 indicated incipient host differentiation between larvae collected from sugarcane or maize. Our estimates of population size changes indicated that a historical bottleneck was associated with a reduction of sugarcane production ≈200 years ago. However, a more recent population expansion was detected (>1950s), associated with agricultural expansion of large crop production into previously unfarmed land. Partial Mantel tests supported our hypothesis of incipient host adaptation, and identified isolation-by-environment (e.g., host plant) in São Paulo and Minas Gerais states, where sugarcane has been traditionally produced in Brazil. The impact of agricultural production on D. saccharalis may continue, as the current population structure may hinder the efficacy of refuge plants in delaying insect resistance evolution to Bt toxin.