Genome-wide identification of miRNAs and target regulatory network in the invasive ectoparasitic mite Varroa destructor.

Varroa destructor is an ectoparasite mite that attacks bees leading to colony disorders worldwide. microRNAs (miRNAs) are key molecules used by eukaryotes to post-transcriptional control of gene expression. Nevertheless, still lack information aboutV. destructor miRNAs and its regulatory networks. Here, we used an integrative strategy to characterize the miRNAs in the V. destructor mite. We identified 310 precursors that give rise to 500 mature miRNAs, which 257 are likely mite-specific elements. miRNAs showed canonical length ranging between 18 and 25 nucleotides and 5' uracil preference. Top 10 elements concentrated over 80% of total miRNA expression, with bantam alone representing ~50%. We also detected non-templated bases in precursor-derived small RNAs, indicative of miRNA post-transcriptional regulatory mechanisms. Finally, we note that conserved miRNAs control similar processes in different organisms, suggesting a conservative role. Altogether, our findings contribute to the better understanding of the mite biology that can assist future studies on varroosis control.

Ceratocystis cacaofunesta differentially modulates the proteome in xylem-enriched tissue of cocoa genotypes with contrasting resistance to Ceratocystis wilt.

MAIN CONCLUSION: Decreased accumulation of polyphenol oxidase, H2O2 accumulation, effective regulation of programmed cell death, and a protein predicted as allergenic can play key roles in cacao defense against Ceratocystis cacaofunesta. Ceratocystis wilt, caused by the fungus Ceratocystis cacaofunesta, has destroyed millions of Theobroma cacao trees in several countries of the Americas. Through proteomics, systems biology, and enzymatic analyses of infected stems, it was possible to infer mechanisms used by resistant (TSH1188) and susceptible (CCN51) cacao genotypes during infection. Protein extraction from xylem-enriched tissue of stems inoculated with the fungus and their controls 1 day after inoculation was carried out, followed by separation through two-dimensional gel electrophoresis and identification by mass spectrometry. Enzyme activity was determined at 1, 3, 7 and 15 days after inoculation. A total of 50 differentially accumulated distinct proteins were identified in the treatments of both genotypes and were classified into 10 different categories. An interaction network between homologous proteins from Arabidospsis thaliana was generated for each genotype, using the STRING database and Cytoscape software. Primary metabolism processes were apparently repressed in both genotypes. The resistance factors suggested for genotype TSH1188 were: H2O2 accumulation, effective regulation of programmed cell death, production of phytoalexins derived from tryptophan and furanocoumarins, and participation of a predicted allergenic protein with probable ribonuclease function inhibiting the germination and propagation of the fungus. In the susceptible genotype, it is possible that its recognition and signaling mechanism through proteins from the SEC14 family is easily overcome by the pathogen. Our results will help to better understand the interaction between cacao and one of its most aggressive pathogens, to create disease control strategies.

Genetic breeding and diversity of the genus Passiflora: progress and perspectives in molecular and genetic studies.

Despite the ecological and economic importance of passion fruit (Passiflora spp.), molecular markers have only recently been utilized in genetic studies of this genus. In addition, both basic genetic researches related to population studies and pre-breeding programs of passion fruit remain scarce for most Passiflora species. Considering the number of Passiflora species and the increasing use of these species as a resource for ornamental, medicinal, and food purposes, the aims of this review are the following: (i) to present the current condition of the passion fruit crop; (ii) to quantify the applications and effects of using molecular markers in studies of Passiflora; (iii) to present the contributions of genetic engineering for passion fruit culture; and (iv) to discuss the progress and perspectives of this research. Thus, the present review aims to summarize and discuss the relationship between historical and current progress on the culture, breeding, and molecular genetics of passion fruit.

Molecular genetic variability of commercial and wild accessions of passion fruit (Passiflora spp.) targeting ex situ conservation and breeding.

Passiflora species are distributed throughout Latin America, and Brazil and Colombia serve as the centers of diversity for this genus. We performed cross-species amplification to evaluate 109 microsatellite loci in 14 Passiflora species and estimated the diversity and genetic structure of Passiflora cincinnata, Passiflora setaceae and Passiflora edulis. A total of 127 accessions, including 85 accessions of P. edulis, a commercial species, and 42 accessions of 13 wild species, were examined. The cross-species amplification was effective for obtaining microsatellite loci (average cross-amplification of 70%). The average number of alleles per locus (five) was relatively low, and the average diversity ranged from 0.52 in P. cincinnata to 0.32 in P. setacea. The Bayesian analyses indicated that the P. cincinnata and P. setacea accessions were distributed into two groups, and the P. edulis accessions were distributed into five groups. Private alleles were identified, and suggestions for core collections are presented. Further collections are necessary, and the information generated may be useful for breeding and conservation.

Development and characterization of microsatellite markers for the wild South American Passiflora cincinnata (Passifloraceae).

PREMISE OF THE STUDY: We developed the first set of microsatellite markers for the passion fruit, Passiflora cincinnata, to provide tools for further study of its genetic diversity and to support current conservation and genetic studies. METHODS AND RESULTS: We used a microsatellite-enriched library approach to isolate and characterize 25 new molecular markers. Seven primer pairs successfully amplified polymorphic microsatellite regions, with an average of five alleles per locus. The mean values of expected and observed levels of heterozygosity were 0.516 and 0.525, respectively. CONCLUSIONS: The microsatellite markers identified in this study may be valuable tools for population genetic studies, and this set of markers also may be useful in the design of a genetic conservation strategy, mating system, and hybridization studies.

Interethnic diversity of NAT2 polymorphisms in Brazilian admixed populations.

BACKGROUND: N-acetyltransferase type 2 (Nat2) is a phase II drug- metabolizing enzyme that plays a key role in the bioactivation of aromatic and heterocyclic amines. Its relevance in drug metabolism and disease susceptibility remains a central theme for pharmacogenetic research, mainly because of its genetic variability among human populations. In fact, the evolutionary and ethnic-specific SNPs on the NAT2 gene remain a focus for the potential discoveries in personalized drug therapy and genetic markers of diseases. Despite the wide characterization of NAT2 SNPs frequency in established ethnic groups, little data are available for highly admixed populations. In this context, five common NAT2 SNPs (G191A, C481T, G590A, A803G and G857A) were investigated in a highly admixed population comprised of Afro-Brazilians, Whites, and Amerindians in northeastern Brazil. Thus, we sought to determine whether the distribution of NAT2 polymorphism is different among these three ethnic groups. RESULTS: Overall, there were no statistically significant differences in the distribution of NAT2 polymorphism when Afro-Brazilian and White groups were compared. Even the allele frequency of 191A, relatively common in African descendents, was not different between the Afro-Brazilian and White groups. However, allele and genotype frequencies of G590A were significantly higher in the Amerindian group than either in the Afro-Brazilian or White groups. Interestingly, a haplotype block between G590A and A803G was verified exclusively among Amerindians. CONCLUSIONS: Our results indicate that ethnic admixture might contribute to a particular pattern of genetic diversity in the NAT2 gene and also offer new insights for the investigation of possible new NAT2 gene-environment effects in admixed populations.

Glutathione s-transferase variants in a brazilian population.

Recent pharmacogenomic studies have revealed significant interethnic differences in glutathione S-transferase (GST) allelic frequencies among various ethnic groups. Therefore, we have investigated GSTM1 (gene deletion), GSTT1 (gene deletion) and GSTP1 (rs1695) polymorphism frequencies in 3 Brazilian ethnic groups (n = 203). GSTM1 and GSTT1 polymorphism analyses were performed by multiplex polymerase chain reaction, and GSTP1 (rs1695) analysis was done by polymerase chain reaction restriction fragment length polymorphism. GSTM1- polymorphism frequency was 33.2%, while GSTT1 null (GSTT1-) was 30.2%. The valine GSTP1*B (rs1695) allele was present in 35.1% subjects, while the heterozygous form (isoleucine/valine) was the most prevalent genotype (46.6%). We found a statistically significant difference in genotype frequency among Amerindians versus Caucasians (p = 0.016) and among Amerindians versus African-Americans (p = 0.033). Considerable frequency variation was found in our study, even when compared with other studies showing phylogeographical heterogeneity to the genes studied in Brazilian populations.

Mapping of a Major QTL for Ceratocystis Wilt Disease in an F1 Population of Theobroma cacao.

Cacao is an important crop, its beans are key raw materials for the chocolate and cosmetic industries. Ceratocystis wilt of cacao (CWC) caused by Ceratocystis cacaofunesta is a lethal disease for the crop. Therefore, the selection of resistant cacao varieties is one of the viable ways to minimize losses in cacao production. In this paper, we described the identification of a major QTL associated with CWC in an F1 mapping population from a cross between a resistant, "TSH 1188," and a susceptible genotype, "CCN 51." A set of 266 trees were genotyped using 3,526 single nucleotide polymorphic markers and then multiple QTL mapping analyses were performed. Two QTLs were identified on chromosomes IV and VI. The major QTL was located at 20 cM from the top position of chromosome VI, accounting for more than 60% of the phenotypic variation. The favorable allele T1, with haplotype GTT, came from the "TSH 1188" parent. It was evident that the haplotype combination T1C2 on chromosome VI was the most significant for resistance, since 93% of resistant trees had this haplotype. The major QTL converged to a genomic region of 739.4 kb that harbored nine candidate genes, including two major classes of resistance genes, which would make them the primary candidates involved in the resistance to CWC. The haplotypes detected are now used to improve the efficiency and precision of the selection of resistant trees in cacao breeding.

Genetic Structure and Molecular Diversity of Cacao Plants Established as Local Varieties for More than Two Centuries: The Genetic History of Cacao Plantations in Bahia, Brazil.

Bahia is the most important cacao-producing state in Brazil, which is currently the sixth-largest country worldwide to produce cacao seeds. In the eighteenth century, the Comum, Pará and Maranhão varieties of cacao were introduced into southern Bahia, and their descendants, which are called 'Bahian cacao' or local Bahian varieties, have been cultivated for over 200 years. Comum plants have been used to start plantations in African countries and extended as far as countries in South Asia and Oceania. In Brazil, two sets of clones selected from Bahian varieties and their mutants, the Agronomic Institute of East (SIAL) and Bahian Cacao Institute (SIC) series, represent the diversity of Bahian cacao in germplasm banks. Because the genetic diversity of Bahian varieties, which is essential for breeding programs, remains unknown, the objective of this work was to assess the genetic structure and diversity of local Bahian varieties collected from farms and germplasm banks. To this end, 30 simple sequence repeat (SSR) markers were used to genotype 279 cacao plants from germplasm and local farms. The results facilitated the identification of 219 cacao plants of Bahian origin, and 51 of these were SIAL or SIC clones. Bahian cacao showed low genetic diversity. It could be verified that SIC and SIAL clones do not represent the true diversity of Bahian cacao, with the greatest amount of diversity found in cacao trees on the farms. Thus, a core collection to aid in prioritizing the plants to be sampled for Bahian cacao diversity is suggested. These results provide information that can be used to conserve Bahian cacao plants and applied in breeding programs to obtain more productive Bahian cacao with superior quality and tolerance to major diseases in tropical cacao plantations worldwide.

New microsatellite markers for wild and commercial species of Passiflora (Passifloraceae) and cross-amplification.

PREMISE OF THE STUDY: We developed the first microsatellites for Passiflora setacea and characterized new sets of markers for P. edulis and P. cincinnata, enabling further genetic diversity studies to support the conservation and breeding of passion fruit species. • METHODS AND RESULTS: We developed 69 microsatellite markers and, in conjunction with assessments of cross-amplification using primers available from the literature, present 43 new polymorphic microsatellite loci for three species of Passiflora. The mean number of alleles per locus was 3.1, and the mean values of the expected and observed levels of heterozygosity were 0.406 and 0.322, respectively. • CONCLUSIONS: These microsatellite markers will be valuable tools for investigating the genetic diversity and population structure of wild and commercial species of passion fruit (Passiflora spp.) and may be useful for developing conservation and improvement strategies by contributing to the understanding of the mating system and hybridization within the genus.

Karyotype, heterochromatin distribution and meiosis of Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae).

Zabrotes subfasciatus (Boh.) has been extensively studied in its agronomic and biochemical aspects due to its importance as a damaging insect to leguminous grains during storage. The few cytogenetic studies published on this species yielded conflicting results. In this study, the karyotype was analyzed in order to accurately describe the chromosome C-banding patterns and meiosis. The brain ganglion at the prepupa and the adult and pupal testes were analyzed. All individuals had 26 chromosomes in both brain ganglion and spermatogonic mitotic metaphases. These chromosomes were classified as follows: the 12th pair and the Y chromosome were telocentric; the X chromosome was acrocentric; the 4th and 5th pairs were submetacentric; and the remaining pairs were all metacentric. One of the members of the 5th pair presented a secondary constriction. All chromosomes presented pericentromeric heterochromatin. The large arms of the pairs 5, 9 and X presented heterochromatin. The X chromosome showed to be heteropyknotic throughout the prophase of the first meiotic division. The subphases of prophase I were atypical and meiosis II was rarely identified. Testes of all males showed a few cells; the bivalents were rod-like shaped in metaphase I. Karyological formulae were 2n = 24 + XX in females and 2n = 24 + XYp and either n = 12 + X or n = 12 + Y in males.

Consistent interethnic differences in the distribution of clinically relevant endothelial nitric oxide synthase genetic polymorphisms.

A maldistribution of endothelial nitric oxide synthase (eNOS) genetic variants may explain differences in NO-mediated effects and response to drugs among black and white subjects. While interethnic differences in the distribution of eNOS genetic variants exist in the American population, it is not known whether such interethnic differences exist in other populations. To test this possibility, we examined the distribution of genetic variants of three clinically relevant eNOS polymorphisms (T(-786)C in the promoter, the VNTR in intron 4, and the Glu298Asp variant in exon 7) in 136 black and 154 white subjects from a Brazilian population, which is very heterogeneous. We also estimated the haplotype frequency and evaluated associations between these variants. The Asp298 variant was more common in whites (32.8%) than in blacks (15.1%) (P < 0.004). Similarly, the C(-786) variant was more common in whites (41.9%) than in blacks (19.5%) (P < 0.0004). However, the 4a variant was more common in blacks (32.0%) than in whites (17.9%) (P < 0.003). The most common predicted haplotype in both ethnic groups combined only wild-type variants. While the second most common haplotype in blacks includes the variant 4a and the wild-type variants for the remaining polymorphisms, the second most common haplotype in whites includes the variants Asp298 and C(-786) and the wild-type variant for polymorphism in intron 4. The marked interethnic differences that we found in Brazilians are very similar to those previously reported in Americans. These findings strongly suggest a consistent difference in the distribution of eNOS genetic variants in blacks compared with whites and indicate that the interethnic differences do not vary with geographic origin.

Karyotype, heterochromatin distribution and meiosis of Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae) / Cariótipo, distribuição da heterocromatina e meiose de Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae)

Zabrotes subfasciatus (Boh.) é estudada intensivamente em termos agronômicos e bioquímicos por causar danos aos grãos de leguminosas armazenados. No entanto, os dados publicados sobre o seu cariótipo são escassos e conflitantes. Assim, o objetivo deste estudo foi descrever o cariótipo e a meiose desse inseto e analisar o padrão de bandas-C de seus cromossomos. Foram analisados os gânglios cerebrais de pré-pupas e os testículos de adultos e pupas com adaptação de uma técnica que permitiu boa qualidade de preparo dos cromossomos dessa espécie. Todos os indivíduos apresentaram 26 cromossomos nas metáfases mitóticas. Esses cromossomos foram classificados em: acrocêntricos (cromossomo X); submetacêntricos (pares 4 e 5); subtelocêntricos (par 12 e cromossomo y); metacêntricos (demais pares). O cromossomo 5 apresentou uma constrição secundária. Todos os cromossomos apresentaram heterocromatina próximo ao centrômero e os cromossomos 5, 9 e X, nos braços longos. O cromossomo X mostrou-se heteropicnótico durante toda a prófase da primeira divisão meiótica. As subfases da prófase I foram pouco distintas e a meiose II de difícil identificação. Os testículos de todos os machos apresentaram poucas células. Os bivalentes apresentaram a forma de bastão na metáfase I. O seu cariótipo constitui-se de 26 cromossomos, sendo as fórmulas cariotípicas 2n = 24 + XX nas fêmeas e 2n = 24 + Xyp e n = 12 + X ou n = 12 + y nos machos.

Zabrotes subfasciatus (Boh.) has been extensively studied in its agronomic and biochemical aspects due to its importance as a damaging insect to leguminous grains during storage. The few cytogenetic studies published on this species yielded conflicting results. In this study, the karyotype was analyzed in order to accurately describe the chromosome C-banding patterns and meiosis. The brain ganglion at the prepupa and the adult and pupal testes were analyzed. All individuals had 26 chromosomes in both brain ganglion and spermatogonic mitotic metaphases. These chromosomes were classified as follows: the 12th pair and the Y chromosome were telocentric; the X chromosome was acrocentric; the 4th and 5th pairs were submetacentric; and the remaining pairs were all metacentric. One of the members of the 5th pair presented a secondary constriction. All chromosomes presented pericentromeric heterochromatin. The large arms of the pairs 5, 9 and X presented heterochromatin. The X chromosome showed to be heteropyknotic throughout the prophase of the first meiotic division. The subphases of prophase I were atypical and meiosis II was rarely identified. Testes of all males showed a few cells; the bivalents were rod-like shaped in metaphase I. Karyological formulae were 2n = 24 + XX in females and 2n = 24 + XYp and either n = 12 + X or n = 12 + Y in males.