Genetic variability of garlic accessions as revealed by agro-morphological traits evaluated under different environments.

The cultivated garlic (Allium sativum L.) displays a wide phenotypic diversity, which is derived from natural mutations and phenotypic plasticity, due to dependence on soil type, moisture, latitude, altitude and cultural practices, leading to a large number of cultivars. This study aimed to evaluate the genetic variability shown by 63 garlic accessions belonging to Instituto Agronômico de Campinas and the Escola Superior de Agricultura "Luiz de Queiroz" germplasm collections. We evaluated ten quantitative characters in experimental trials conducted under two localities of the State of São Paulo: Monte Alegre do Sul and Piracicaba, during the agricultural year of 2007, in a randomized blocks design with five replications. The Mahalanobis distance was used to measure genetic dissimilarities. The UPGMA method and Tocher's method were used as clustering procedures. Results indicated significant variation among accessions (P < 0.01) for all evaluated characters, except for the percentage of secondary bulb growth in MAS, indicating the existence of genetic variation for bulb production, and germplasm evaluation considering different environments is more reliable for the characterization of the genotypic variability among garlic accessions, since it diminishes the environmental effects in the clustering of genotypes.

Pan-American Similarities in Genetic Structures of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) With Implications for Hybridization.

The genus Helicoverpa (Lepidoptera: Noctuidae) includes phytophagous and polyphagous agricultural insect pests. In the Americas, a native pest, Helicoverpa zea (Boddie), and an invasive pest, Helicoverpa armigera (Hübner), are causing severe damage in vegetable and agronomic crops. The population structure of both species in South America is poorly understood, and the phylogenetic relatedness of H. armigera and H. zea suggests natural interspecific gene flow between these species. Using microsatellite loci, we investigated: 1) the genetic diversity and gene flow of H. armigera specimens from Brazil; 2) the genetic diversity and gene flow between H. zea specimens from Brazil and the United States; and 3) the possibility of interspecific gene flow and the frequency of putative hybrids in Brazil. We detected high intraspecific gene flow among populations collected in the same country. However, there is a geographic limit to gene flow among H. zea individuals from South and North America. Pairwise Fst and private alleles showed that H. armigera is more similar to H. zea from Brazil than H. zea from the United States. A comparative STRUCTURE analysis suggests natural hybridization between H. armigera and H. zea in Brazil. High gene flow and natural hybridization are key traits to population adaptation in new and disturbed environments, which can influence the management of these pests in the American continent.

Chloroplast diversity of Casearia grandiflora in the Cerrado of Piauí State.

Casearia grandiflora (Salicaceae) is a typical Cerrado species adapted to disturbed environments, making it useful for restoration projects. Knowledge of genetic diversity is important for establishing conservation strategies for this species. This study aimed to compare chloroplast haplotype diversity and structure of C. grandiflora, under the assumption that protected areas hold greater genetic diversity than disturbed areas. The populations studied are from Parque Nacional de Sete Cidades Conservation Unit and from the surroundings of the city of Cocal de Telha, both located in the State of Piauí. Molecular analyses were performed with seven chloroplast microsatellite loci. The number of private haplotypes and haplotype diversity were higher in the conservation unit, which reinforces the importance of these areas in maintaining biodiversity. Analysis of molecular variance showed that most of the genetic variation is found within populations, with a moderate divergence between them (FST = 0.14). The Bayesian analysis and discriminant analysis of principal components suggested that populations are not structured, revealing that a set of individuals from Parque Nacional de Sete Cidades were more divergent within populations than between them. Since literature has little information on C. grandiflora, the results of this study provide important contribution to a better understanding of the specie's genetic diversity.

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.

Restriction site associated DNA (RAD) for de novo sequencing and marker discovery in sugarcane borer, Diatraea saccharalis Fab. (Lepidoptera: Crambidae).

We present the development of a genomic library using RADseq (restriction site associated DNA sequencing) protocol for marker discovery that can be applied on evolutionary studies of the sugarcane borer Diatraea saccharalis, an important South American insect pest. A RADtag protocol combined with Illumina paired-end sequencing allowed de novo discovery of 12 811 SNPs and a high-quality assembly of 122.8M paired-end reads from six individuals, representing 40 Gb of sequencing data. Approximately 1.7 Mb of the sugarcane borer genome distributed over 5289 minicontigs were obtained upon assembly of second reads from first reads RADtag loci where at least one SNP was discovered and genotyped. Minicontig lengths ranged from 200 to 611 bp and were used for functional annotation and microsatellite discovery. These markers will be used in future studies to understand gene flow and adaptation to host plants and control tactics.

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.

Reproductive isolation between two populations of Diatraea saccharalis (F.) (Lepidoptera: Crambidae) from different host plant species and regions in Argentina.

The sugarcane borer, Diatraea saccharalis (F.), has a widespread distribution throughout the Western Hemisphere and is a pest of many crop plants including sugarcane, corn, sorghum and rice. The use of Bacillus thuringiensis (Bt) corn has been the primary tool for managing this species in corn fields. Sugarcane borer control has been recently threatened by observations of susceptibility and/or resistance to certain varieties of Bt corn and the protein used in many newer varieties. This has led to increased interest in understanding sugarcane borer genetic diversity and gene flow within and among its populations and the consequent exchange of alleles between geographically distant populations. The objective of this study was to examine reproductive compatibility between host-associated geographic populations of D. saccharalis in Argentina and to determine whether this pest represents a complex of host-associated cryptic species rather than a wide ranging generalist species. Intra and inter-population crosses revealed that D. saccharalis populations from the northwestern and Pampas regions presented evidence of prezygotic and postzygotic incompatibility. Such a result is likely to be the product of an interruption of gene flow produced by either geographic or host plant associated isolation, suggesting that Tucumán (northwestern) and Buenos Aires (Pampas) populations of D. saccharalis are a distinct genotype and possibly an incipient species.

Molecular characterization of patchouli (Pogostemon spp) germplasm.

Patchouli [Pogostemon cablin (Blanco) Benth.] is an aromatic, herbaceous plant belonging to the Lamiaceae family native to Southeast Asia. Its leaves produce an essential oil regularly used by the perfume and cosmetics industries. However, since patchouli from the Philippines and India were described and named Pogostemon patchouli, there has been a divergence in the identity of these species. The objective of the current study was to study the genetic diversity of patchouli accessions in the Active Germplasm Bank of Universidade Federal de Sergipe using microsatellite and inter simple sequence repeat markers. The results of both types of molecular markers showed that there are two well-defined clusters of accessions that harbor exclusive alleles. It was observed that these two clusters are genetically distant, suggesting that they belong to two different species. Based on the results, two accessions were classified as Pogostemon heyneanus and the remaining accessions were classified as P. cablin.

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.

Populations of Erythrina velutina Willd. at risk of extinction.

The goal of this study was to characterize the structure of two natural populations of the coral tree using RAPD and ISSR markers. The study evaluated all individuals in two different areas in the northeastern region of Brazil: the first was in the riparian area, 10 km x 100 m along the edge of the lower São Francisco River, and the second was in the municipality of Pinhão, in a semiarid region between the municipalities of Neópolis and Santana do São Francisco. We used all the coral trees present in those two areas (37 individuals). The results of the RAPD and ISSR markers were highly congruent, supporting the reliability of the techniques used. Similarity was estimated using the Jaccard arithmetic complement index. A dendrogram was constructed using the unweighted pair group method with arithmetic mean cluster algorithm, and the robustness of the data was bootstrapped with 5000 replicates. A principal coordinate analysis was performed on the basis of Jaccard coefficients. The total genetic variation observed was 21%, corresponding to the variation between the populations, and 79% of the variation was observed within the populations.

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.

New loci of Lychnophora ericoides and transferability to Lychnophora pinaster, endangered medicinal species from Brazil.

Lychnophora ericoides and Lychnophora pinaster are species used in popular medicine as analgesic or anti-inflammatory agents to treat contusions, rheumatism, and insect bites. In this study, 21 simple sequence repeat loci of L. ericoides were developed and transferred to L. pinaster. Three populations of L. ericoides and 2 populations of L. pinaster were evaluated; they were collected in the State of Minas Gerais. Population parameters were estimated, and the mean values of observed and expected heterozygosity were 0.297 and 0.408 (L. ericoides) and 0.228 and 0.310 (L. pinaster), respectively. Greater genetic variability was observed within populations than between populations of L. ericoides (62 and 37%) and L. pinaster (97 and 2.8%). These results provide information for genetic conservation and taxonomic studies of these endangered species.

Isolation and characterization of microsatellite loci from the tick Amblyomma aureolatum (Acari: Ixodidae).

Amblyomma aureolatum (Pallas) is the main vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever. This disease is the most lethal human spotted fever rickettsiosis in the world. Microsatellite loci were isolated from a dinucleotide-enriched library produced from A. aureolatum sampled in Southeastern Brazil. Eight polymorphic microsatellites were further characterized among 38 individuals sampled from São Paulo metropolitan region. The number of observed alleles ranged from 2 to 9, observed heterozygosity was 0.184-0.647, and expected heterozygosity was 0.251-0.747. Cross-species amplifications suggested that these loci will be useful for other Amblyomma 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.

Development and characterization of novel microsatellite markers in Hyptis pectinata (Lamiaceae).

A microsatellite-enriched library was constructed and a set of 19 SSR markers were developed to characterize a germplasm collection of Hyptis pectinata (L.) Poit., maintained at the Universidade Federal de Sergipe (UFS). Fifteen markers of 19 ranged from moderately to highly polymorphic. A total of 113 alleles were identified, with a mean of 7.52 alleles per locus. The mean HO and HE were 0.582 and 0.657, respectively. The primers developed were efficient tools for accessing the genetic diversity of the germplasm collection analyzed and may also be useful for other studies involving this species and other species in the genus Hyptis.

Isolation and characterization of microsatellite loci for Bixa orellana, an important source of natural dyes.

Annatto (Bixa orellana) is a plant native from the American continental tropical zone. The seeds are used to produce a carotenoid-based yellow to orange food coloring. Microsatellite markers were developed for the Brazilian native species Bixa orellana to describe its genetic diversity and structure as well as to support conservation studies. Twenty-five microsatellite loci were isolated and characterized using an enriched genomic library. Ten loci were polymorphic in the 50 accessions sampled in this study, while 15 were considered monomorphic. The mean number of alleles per locus was 3.8, ranging from 2 to 6 alleles per locus. Mean values for the observed and expected heterozygosities were 0.541 (ranging from 0 to 0.658) and 0.639 (ranging from 0.422 to 0.787), respectively. All markers described in this study will be useful in further studies evaluating the genetic diversity, population dynamics, and conservation genetics of Bixa orellana.

A modified acidic approach for DNA extraction from plant species containing high levels of secondary metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Isolation and characterization of microsatellites for the yam Dioscorea cayenensis (Dioscoreaceae) and cross-amplification in D. rotundata.

Dioscorea cayenensis and Dioscorea rotundata are among the most important yam species for the humid and sub-humid tropics. We isolated nine polymorphic microsatellite markers using a microsatellite-enriched genomic library technique. The nine primer pairs were validated in 22 D. cayenensis accessions, and were tested for transferability in 26 D. rotundata accessions. The number of bands ranged from 2 to 4, with a mean of 3.11. D. cayenensis gave primer polymorphism information content values ranging from 0.37 to 0.62, while for D. rotundata the values ranged from 0.15 to 0.66. The D parameter in D. cayenensis ranged from 0.14 to 0.40, while in D. rotundata it ranged from 0.05 to 0.34. These SSR markers will be useful to characterize genetic diversity in D. cayenensis and D. rotundata accessions.

Microsatellite markers for the Cabreúva tree, Myroxylon peruiferum (Fabaceae), an endangered medicinal species from the Brazilian Atlantic Forest.

The Cabreúva tree, Myroxylon peruiferum, is an endangered tropical species from Brazil used in forest restoration projects. It is known for its medicinal properties. Eleven microsatellite markers were developed for this species, from a microsatellite-enriched library. Nine of these markers, characterized in 30 individuals from a semideciduous forest remnant population in southeast Brazil, were polymorphic, with allele numbers ranging from 2 to 8 per locus; expected and observed heterozygosities ranged from 0.103 to 0.757 and 0.107 to 0.704, respectively. One locus (Mpe-C04) showed significant deviation from Hardy-Weinberg equilibrium, probably due to null alleles. Two other loci (Mpe-E09 and Mpe-H07) were monomorphic in this population. These microsatellite loci should be useful for future population genetic studies of this species.