Phylogenomic and expression analysis of Colossoma macropomum cyp19a1a and cyp19a1b and their non-classical role in tambaqui sex differentiation.

The genes coding for Cytochrome P450 aromatase (cyp19a1a and cyp19a1b) and estrogen (E2) receptors (esr1, esr2a and esr2b) play a conserved role in ovarian differentiation and development among teleosts. Classically, the "gonad form" of aromatase, coded by the cyp19a1a, is responsible for the ovarian differentiation in genetic females via ligation and activation of the Esr, which mediates the endocrine and exocrine signaling to allow or block the establishment of the feminine phenotype. However, in neotropical species, studies on the molecular and endocrine processes involved in gonad differentiation as well as on the effects of sex modulators are recent and scarce. In this study, we combined in silico analysis, real-time quantitative PCR (qPCR) assay and quantification of E2 plasma levels of differentiating tambaqui (Colossoma macropomum) to unveil the roles of the paralogs cypa19a1a and cyp19a1b during sex differentiation. Although the synteny of each gene is very conserved among characids, the genomic environment displays striking differences in comparison to model teleost species, with many rearrangements in cyp19a1a and cyp19a1b adjacencies and transposable element traces in both regulatory regions. The high dissimilarity (DI) of SF-1 binding motifs in cyp19a1a (DI = 10.06 to 14.90 %) and cyp19a1b (DI = 8.41 to 13.50 %) regulatory region, respectively, may reflect in an alternative pathway in tambaqui. Indeed, while low transcription of cyp19a1a was detected prior to sex differentiation, the expression of cyp19a1b and esr2a presented a large variation at this phase, which could be associated with sex-specific differential expression. Histological analysis revealed that anti-estradiol treatments did not affect gonadal sex ratios, although Fadrozole (50 mg kg-1 of food) reduced E2 plasma levels (p < 0,005) as well cyp19a1a transcription; and tamoxifen (200 mg kg-1 of food) down regulated both cyp19a1a and cyp19a1b but did not influence E2 levels. Altogether, our results bring into light new insights about the evolutionary fate of cyp19a1 paralogs in neotropical fish, which may have generated uncommon roles for the gonadal and brain forms of cyp19a1 genes and the unexpected lack of effect of endocrine disruptors on tambaqui sexual differentiation.

Antifungal activity of poly(ε-caprolactone) nanoparticles incorporated with Eucalyptus essential oils against Hemileia vastatrix.

Coffee (Coffea L.) is one of the main crops produced globally. Its contamination by the fungus Hemileia vastatrix Berkeley and Broome has been economically detrimental for producers. The objective of this work was to extract and characterize the essential oils from Eucalyptus citriodora Hook, Eucalyptus camaldulensis Dehn and Eucalyptus grandis Hill ex Maiden, produce and characterize nanoparticles containing these essential oils and evaluate the in vivo and in vitro antifungal activity of free and nanoencapsulated essential oils. The principal constituent of the essential oil from E. citriodora was citronellal; that from E. grandis was α-pinene; and that from E. camaldulensis was 1,8-cineol. The in vitro antifungal activity against the fungus H. vastatrix was 100% at a concentration of 1000 µl l-1 for all the oils and nanoparticles containing these natural products. The sizes of the nanoparticles produced with the essential oils from E. citriodora, E. camaldulensis and E. grandis were 402·13 nm, 275·33 nm and 328·5 nm, respectively, with surface charges of -11·8 mV, -9·24 mV and - 6·76 mV, respectively. Fourier transform infrared analyses proved that the encapsulation of essential oils occurred in the polymeric matrix of poly(ε-caprolactone). The incorporation of essential oils into biodegradable poly(ε-caprolactone) nanoparticles increased their efficiency as biofungicides in the fight against coffee rust, decreasing the severity of the disease by up to 90·75% after treatment with the nanoparticles containing the essential oil from E. grandis.

Bactericidal and antioxidant effects of essential oils from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus.

The extraction and characterization of the essential oils (EO) from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus and the determination of their antibacterial and antioxidant activities were achieved. The EO were identified by gas chromatography/mass spectrometry and quantified by gas chromatography using a flame ionization detector. The antibacterial potential against Escherichia coli and Staphylococcus aureus was evaluated by cell susceptibility assays and by scanning electron microscopy. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl assay, by ß-carotene bleaching and by determining the reducing power. Borneol (36·18%), γ-terpineol (12·66%) and carvacrol (11·07%) were the principal components in the EO from S. montana, and sabinene (49·23%) and α-pinene (13·81%) were found in the EO from M. fragrans. Geranial (59·66%) and neral (38·98%) isomers were the only major components in the EO from C. flexuosus. The EO from S. montana was effective against E. coli, with minimum inhibitory and bactericidal concentrations (MIC and MBC) of 6·25 µl ml-1 , whereas bactericidal potential against both was observed for the EO from M. fragrans; MIC = 6·25 µl ml-1 for S. aureus and MBC = 12·5 µl ml-1 for E. coli. A significant protective role on lipid substrates in the ß-carotene bleaching assay was seen for the EO from S. montana and M. fragrans. Overall, such EO can be promising agents against pathogenic bacteria and for protecting biomolecules during oxidative stress.

Origin and population structure of Brazilian hair sheep breeds.

Brazilian hair sheep constitute a genetic diversity hotspot. These animals are found in the harsh environments of the Brazilian Northwest (semi-arid) region. Genotypes (50K SNP chip) from seven Brazilian sheep breeds (five hair and two coarse wool types) and 87 worldwide breeds were used to test for population structure, admixture and genetic diversity. Moreover, phylogenetic trees evaluating migration events between genetic groups were built. Brazilian Somali, a fat-tailed breed, had a close relationship with East African breeds and clustered distinctly from other Brazilian breeds. Brazilian Blackbelly and Barbados Blackbelly had a close relationship. The Morada Nova breed did not show close relationships with European or African breeds, revealing a single migration event from an Algerian hair breed. Brazilian Fat-tail and Morada Nova share a common ancestor, but the former showed introgressions from Brazilian Somali and Afrikaner breeds, explaining the fat-tail phenotype. The Santa Inês breed received a substantial contribution from Brazilian Bergamasca and showed an admixed origin with recent introgressions from other breeds, mainly from Suffolk. Furthermore, Brazilian Somali and Brazilian Fat-tail are the most endangered sheep genetic resources in Brazil and should be the focus for ex situ conservation programs. In conclusion, Brazilian hair sheep show an African origin and are characterized by diverse genetic composition, reinforcing the need for conservation of these genetic resources, and at the same time, this highly diverse group has variability that can be used in breeding programs.

Autoregressive single-step test-day model for genomic evaluations of Portuguese Holstein cattle.

The multiple-lactation autoregressive test-day (AR) model is the adopted model for the national genetic evaluation of dairy cattle in Portugal. Under this model, animals' permanent environment effects are assumed to follow a first-order autoregressive process over the long (auto-correlations between parities) and short (auto-correlations between test-days within lactation) terms. Given the relevance of genomic prediction in dairy cattle, it is essential to include marker information in national genetic evaluations. In this context, we aimed to evaluate the feasibility of applying the single-step genomic (G)BLUP to analyze milk yield using the AR model in Portuguese Holstein cattle. In total, 11,434,294 test-day records from the first 3 lactations collected between 1994 and 2017 and 1,071 genotyped bulls were used in this study. Rank correlations and differences in reliability among bulls were used to compare the performance of the traditional (A-AR) and single-step (H-AR) models. These 2 modeling approaches were also applied to reduced data sets with records truncated after 2012 (deleting daughters of tested bulls) to evaluate the predictive ability of the H-AR. Validation scenarios were proposed, taking into account young and proven bulls. Average EBV reliabilities, empirical reliabilities, and genetic trends predicted from the complete and reduced data sets were used to validate the genomic evaluation. Average EBV reliabilities for H-AR (A-AR) using the complete data set were 0.52 (0.16) and 0.72 (0.62) for genotyped bulls with no daughters and bulls with 1 to 9 daughters, respectively. These results showed an increase in EBV reliabilities of 0.10 to 0.36 when genomic information was included, corresponding to a reduction of up to 43% in prediction error variance. Considering the 3 validation scenarios, the inclusion of genomic information improved the average EBV reliability in the reduced data set, which ranged, on average, from 0.16 to 0.26, indicating an increase in the predictive ability. Similarly, empirical reliability increased by up to 0.08 between validation tests. The H-AR outperformed A-AR in terms of genetic trends when unproven genotyped bulls were included. The results suggest that the single-step GBLUP AR model is feasible and may be applied to national Portuguese genetic evaluations for milk yield.

A fine structure genetic analysis evaluating ecoregional adaptability of a Bos taurus breed (Hereford).

Ecoregional differences contribute to genetic environmental interactions and impact animal performance. These differences may become more important under climate change scenarios. Utilizing genetic diversity within a species to address such problems has not been fully explored. In this study Hereford cattle were genotyped with 50K Bead Chip or 770K Bovine Bead Chip to test the existence of genetic structure in five U.S. ecoregions characterized by precipitation, temperature and humidity and designated: cool arid (CA), cool humid (CH), transition zone (TZ), warm arid (WA), and warm humid (WH). SNP data were analyzed in three sequential analyses. Broad genetic structure was evaluated with STRUCTURE, and ADMIXTURE software using 14,312 SNPs after passing quality control variables. The second analysis was performed using principal coordinate analysis with 66 Tag SNPs associated in the literature with various aspects of environmental stressors (e.g., heat tolerance) or production (e.g., milk production). In the third analysis TreeSelect was used with the 66 SNPs to evaluate if ecoregional allelic frequencies deviated from a central frequency and by so doing are indicative of directional selection. The three analyses suggested subpopulation structures associated with ecoregions from where animals were derived. ADMIXTURE and PCA results illustrated the importance of temperature and humidity and confirm subpopulation assignments. Comparisons of allele frequencies with TreeSelect showed ecoregion differences, in particular the divergence between arid and humid regions. Patterns of genetic variability obtained by medium and high density SNP chips can be used to acclimatize a temperately derived breed to various ecoregions. As climate change becomes an important factor in cattle production, this study should be used as a proof of concept to review future breeding and conservation schemes aimed at adaptation to climatic events.

Genomic prediction for tick resistance in Braford and Hereford cattle.

One of the main animal health problems in tropical and subtropical cattle production is the bovine tick, which causes decreased performance, hide devaluation, increased production costs with acaricide treatments, and transmission of infectious diseases. This study investigated the utility of genomic prediction as a tool to select Braford (BO) and Hereford (HH) cattle resistant to ticks. The accuracy and bias of different methods for direct and blended genomic prediction was assessed using 10,673 tick counts obtained from 3,435 BO and 928 HH cattle belonging to the Delta G Connection breeding program. A subset of 2,803 BO and 652 HH samples were genotyped and 41,045 markers remained after quality control. Log transformed records were adjusted by a pedigree repeatability model to estimate variance components, genetic parameters, and breeding values (EBV) and subsequently used to obtain deregressed EBV. Estimated heritability and repeatability for tick counts were 0.19 ± 0.03 and 0.29 ± 0.01, respectively. Data were split into 5 subsets using k-means and random clustering for cross-validation of genomic predictions. Depending on the method, direct genomic value (DGV) prediction accuracies ranged from 0.35 with Bayes least absolute shrinkage and selection operator (LASSO) to 0.39 with BayesB for k-means clustering and between 0.42 with BayesLASSO and 0.45 with BayesC for random clustering. All genomic methods were superior to pedigree BLUP (PBLUP) accuracies of 0.26 for k-means and 0.29 for random groups, with highest accuracy gains obtained with BayesB (39%) for k-means and BayesC (55%) for random groups. Blending of historical phenotypic and pedigree information by different methods further increased DGV accuracies by values between 0.03 and 0.05 for direct prediction methods. However, highest accuracy was observed with single-step genomic BLUP with values of 0.48 for -means and 0.56, which represent, respectively, 84 and 93% improvement over PBLUP. Observed random clustering cross-validation breed-specific accuracies ranged between 0.29 and 0.36 for HH and between 0.55 and 0.61 for BO, depending on the blending method. These moderately high values for BO demonstrate that genomic predictions could be used as a practical tool to improve genetic resistance to ticks and in the development of resistant lines of this breed. For HH, accuracies are still in the low to moderate side and this breed training population needs to be increased before genomic selection could be reliably applied to improve tick resistance.

Genetic diversity and population structure of different varieties of Morada Nova hair sheep from Brazil.

The aim of this study was to analyze genetic diversity and population structure among varieties of White (N = 40), Red (N = 32), and Black (N = 31) Morada Nova hair sheep from flocks in the northeastern Brazilian semiarid region. Fifteen nuclear microsatellite markers and two regions of mitochondrial DNA were used. The intra-population analysis demonstrated that the White variety had higher diversity, while the Red variety had the lowest values. The Bayesian analysis to assess the genetic population structure allowed differentiation between White, Red, and Black varieties, and revealed a tendency towards sub-structuring in the White variety flocks from the States of Ceará and Paraíba. The results of analyses of molecular variance showed that the greatest genetic structure was found when comparing flocks rather than varieties (8.59 vs 6.64% of the total variation, P < 0.001). Based on genetic distance, Dtl, both the dendrogram analysis and the principal coordinate analysis showed the formation of two main groups: one composed of White and another of Black and Red individuals. Five and two haplotypes were found for the D-loop region and the ND5 gene, respectively. A haplotype unique to the Red variety was found in the D-loop region and a variety haplotype unique to the Black variety was found in the ND5 gene; however, these frequencies were low and therefore require further validation. These results support the existence of substantial differences between the Red and White varieties and should be used as separate genetic resources and to improve conservation programs.

Bulked segregant analysis of the pirarucu (Arapaima gigas) genome for identification of sex-specific molecular markers.

Arapaima gigas (Osteoglossidae) is one of the largest fish species in the Amazon Basin, attaining lengths of over 2.5 m and weights of over 100 kg. Its flesh is prized, and it has great potential for production in aquaculture systems. However, live pirarucu cannot be reliably sexed visually, even after sexual development, since this species does not have clear external sexual dimorphism. Simple and inexpensive methods for sexing immature pirarucu based on DNA markers would facilitate production of this species in commercial operations. We analyzed A. gigas male and female DNA pools with 566 RAPD primers, generating 2609 fragments, with an estimated 1341 segregating polymorphic markers, and an estimated average spacing of 714 kb, which corresponds to less than 0.1% of the species' genome. Two putative sex-specific fragments were initially identified in bulked samples; but they were not confirmed in a study of individual male and female samples. We suggest that A. gigas has developed a non-chromosomal system of sex determination or, alternatively, that the species has undergone a recent loss of the chromosome carrying the sex-determining locus.

Incidence of bovine leukocyte adhesion deficiency, complex vertebral malformation, and deficiency of uridine-5-monophosphate synthase carriers in Brazilian Girolando cattle.

Among the various hereditary diseases that have been widely studied in dairy cattle, bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine-5-monophosphate synthase (DUMPS), and complex vertebral malformation (CVM) are noteworthy because of their high impact on overall herd productivity as a consequence of increased calf mortality. The aim of this study was to verify the frequency of carriers of BLAD, CVM, and DUMPS mutant alleles in cows and bulls from the National Girolando Progeny Test carried out in Brazil by using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR assays. A total of 777 animals were genotyped for BLAD, 783 for CVM, and 122 for DUMPS. The frequencies of carriers for BLAD and CVM were 0.77 and 1.53%, respectively, whereas no carriers of DUMPS were observed.

Single nucleotide polymorphisms in the bovine genome are associated with the number of oocytes collected during ovum pick up.

The number of follicles recruited in each estrous cycle has gained practical importance in artificial reproductive technology, as it determines the oocyte yield from ultrasound-guided ovum pickup for in vitro embryo production. We aimed to identify single nucleotide polymorphisms (SNPs) in bovine genes related to reproductive physiology and evaluate the association between the candidate SNPs and the number of oocytes collected from ultrasound-guided ovum pickup. We sequenced genomic segments of GDF9, FGF8, FGF10 and BMPR2 and identified seventeen SNPs in the Bos taurus and Bos indicus breeds. Two SNPs cause amino acid changes in the proteins GDF9 and FGF8. Three SNPs in GDF9, FGF8 and BMPR2 were genotyped in 217 Nelore cows (B. indicus), while two previously identified mutations in LHCGR and mitochondrial DNA (mtDNA) were genotyped in the same group. The polymorphisms in GDF9, FGF8, BMRP2 and LHCGR were significantly associated (P<0.01) with the number of oocytes collected by ovum pickup, whereas the SNP in the mtDNA was not. In addition, we estimated an allelic substitution effect of 1.13±0.01 (P<0.01) oocytes for the SNP in the FGF8 gene. The results we report herein provide further evidence to support the hypothesis that genetic variability is an important component of the number of antral follicles in the bovine ovary.

Estimation of taurindicine hybridization of American Zebu cattle in Brazil.

Our objective was to estimate Bos primigenius taurus introgression in American Zebu cattle. One hundred and four American Zebu (Nellore) cattle were submitted to mtDNA, microsatellite and satellite analysis. Twenty-three alleles were detected in microsatellite analysis, averaging 4.6 ± 1.82/locus. Variance component comparisons of microsatellite allele sizes allowed the construction of two clusters separating taurus and indicus. No significant variation was observed when indicus and taurus mtDNA were compared. Three possible genotypes of 1711b satellite DNA were identified. All European animals showed the same restriction pattern, suggesting a Zebu-specific restriction pattern. The frequencies of B. primigenius indicus-specific microsatellite alleles and 1711b satellite DNA restriction patterns lead to an estimate of 14% taurine contribution in purebred Nellore.

PRNP haplotype and genotype frequencies in Brazilian sheep: issues for conservation and breeding programs.

Polymorphisms of PRNP gene have been strongly correlated to the susceptibility/resistance to scrapie in sheep. Variants at the coding positions 136, 154 and 171 have been the most frequently associated to susceptibility to classical scrapie. The aim of this study was to estimate PRNP haplotype and genotype frequencies in a sample of 1400 sheep from 13 different breeds that are representative of the main production regions in Brazil. A total of four different alleles (ARR, ARQ, AHQ and VRQ) and nine genotypes were observed at different frequencies among the investigated breeds. There were distinct patterns of allelic distribution between naturalized and commercial/specialized breeds and different geographic regions. These results will influence the development and management of breeding and conservation programs and will help to develop Brazilian efforts to avoid scrapie epidemics.

Adaptation of a low-cost medium-throughput genotyping system for ovine prion protein gene polymorphims associated with scrapie.

Resistance and susceptibility to scrapie in sheep have been associated with SNPs located at codons 136, 154 and 171 of the prion protein (PRNP) gene. Many countries have sheep breeding programs selecting for resistance to scrapie based on the genotyping of these SNPs. We adapted a fast and robust method for genotyping sheep flocks for these polymorphisms, with reduced costs. Ninety-six samples were genotyped using an adapted SNaPshot PRNP assay, and the results were checked by resequencing. The results showed 100% concordance, using a method that reduces genotyping costs by 70%, by reducing reagent concentrations in the three main steps of the assay (amplicon purification, base extension and final cleanup). This cost reduction should contribute to the development of selection criteria based on PRNP genotyping in countries where assay costs are an important limiting factor.

Mapping MHC genes in river buffalo.

The major histocompatibility complex (MHC) contains a set of genes necessary for antigen presentation in the immune system. This gene dense and polymorphic region of the mammalian genome is of considerable interest due to the role of MHC genes in immune function and animal health. Previous cytogenetic studies have indicated that the MHC in river buffalo resides on the short arm of chromosome 2 (BBU2). A 5000-rad radiation hybrid mapping panel was recently generated to enable construction of a whole genome map of river buffalo. To this end, the aims of this project were to elucidate the general organization of the MHC on BBU2, and to compare gene order within this region to the MHC in cattle. PCR primers were selected from the bovine gene map and used with the BBURH5000 panel to map a set of ten MHC class II genes in river buffalo. Analysis indicates that these genes fall into two linkage groups, consistent with organization of the MHC in cattle. This comparison of buffalo and bovine MHC gene order provides the first insight into the organization of the MHC on river buffalo chromosome 2.

Physical mapping of genes in the porcine ovarian transcriptome.

Reproductive efficiency and associated traits are of major economic importance to the swine industry and have been more difficult to improve genetically than other production traits. Integration of phenotypical data with gene mapping and expression studies provides a powerful approach for dissection of the genetic basis regulating complex traits. We developed a total of 101 polymerase chain reaction-based markers, representing 91 unique genes, for expressed sequence tags previously reported to be putatively differentially expressed in the porcine ovarian transcriptome of a swine line selected on an index of high ovulation rate and embryonic survival. These were subsequently used in physical mapping experiments with a porcine radiation hybrid and somatic cell hybrid panels. Our results increased the information content of the porcine physical map useful for comparative mapping by c. 10%. Moreover, the mapped genes are likely to be biologically relevant to the molecular mechanisms that control ovulation rate in the pig. A total of 12 differentially expressed genes were mapped to regions previously reported to contain quantitative trait loci affecting swine ovulation rate.

Analysis of expressed sequence tags from Musa acuminata ssp. burmannicoides, var. Calcutta 4 (AA) leaves submitted to temperature stresses.

In order to discover genes expressed in leaves of Musa acuminata ssp. burmannicoides var. Calcutta 4 (AA), from plants submitted to temperature stress, we produced and characterized two full-length enriched cDNA libraries. Total RNA from plants subjected to temperatures ranging from 5 degrees C to 25 degrees C and from 25 degrees C to 45 degrees C was used to produce a COLD and a HOT cDNA library, respectively. We sequenced 1,440 clones from each library. Following quality analysis and vector trimming, we assembled 2,286 sequences from both libraries into 1,019 putative transcripts, consisting of 217 clusters and 802 singletons, which we denoted Musa acuminata assembled expressed sequence tagged (EST) sequences (MaAES). Of these MaAES, 22.87% showed no matches with existing sequences in public databases. A global analysis of the MaAES data set indicated that 10% of the sequenced cDNAs are present in both cDNA libraries, while 42% and 48% are present only in the COLD or in the HOT libraries, respectively. Annotation of the MaAES data set categorized them into 22 functional classes. Of the 2,286 high-quality sequences, 715 (31.28%) originated from full-length cDNA clones and resulted in a set of 149 genes.

Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.

Genome activation and developmental block in bovine embryos.

The ultimate goal of in vitro embryo culture systems is to perfectly mimic the condition of oocyte maturation, fertilization and embryo development. These systems are far more complex than standard in vitro cell culture because of the various environments through which the gametes and embryos pass during in vivo development. Improvement of the medium and other culture conditions has allowed for full development of a percentage of the fertilized oocytes but the great majority of bovine zygotes stop developing within a few cell cycles after initiating cleavage. This developmental block arises in the bovine embryo at the eight-cell-stage and is likely correlated with the cytoplasmic quality of the oocyte. Oocytes harbor all mRNAs and proteins needed to reach the fourth or fifth cell cycle, however, embryos that fail to transcribe their own genome fail to further develop. In this article, we review some of the advances in developmental block knowledge and describe a possible role of active embryo transcription that drives incompetent embryos to block and death.