N-acetylcysteine absorption and its potential dual effect improve fitness and fruit yield in Xylella fastidiosa infected plants.

BACKGROUND: Xylella fastidiosa is a multi-host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N-acetylcysteine (NAC), a low-cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N-NAC we proved that this analogue was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60-fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28-fold of the daily sap flow compared to health plants. In addition, NAC-treated citrus variegated chlorosis (CVC) plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed to the increase in fruit yield of CVC-diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants, but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field-based evidence supports the viability of NAC as a sustainable agricultural application. © 2024 Society of Chemical Industry.

Identification of copy number variations in the genome of Dairy Gir cattle.

Studying structural variants that can control complex traits is relevant for dairy cattle production, especially for animals that are tolerant to breeding conditions in the tropics, such as the Dairy Gir cattle. This study identified and characterized high confidence copy number variation regions (CNVR) in the Gir breed genome. A total of 38 animals were whole-genome sequenced, and 566 individuals were genotyped with a high-density SNP panel, among which 36 animals had both sequencing and SNP genotyping data available. Two sets of high confidence CNVR were established: one based on common CNV identified in the studied population (CNVR_POP), and another with CNV identified in sires with both sequence and SNP genotyping data available (CNVR_ANI). We found 10 CNVR_POP and 45 CNVR_ANI, which covered 1.05 Mb and 4.4 Mb of the bovine genome, respectively. Merging these CNV sets for functional analysis resulted in 48 unique high confidence CNVR. The overlapping genes were previously related to embryonic mortality, environmental adaptation, evolutionary process, immune response, longevity, mammary gland, resistance to gastrointestinal parasites, and stimuli recognition, among others. Our results contribute to a better understanding of the Gir breed genome. Moreover, the CNV identified in this study can potentially affect genes related to complex traits, such as production, health, and reproduction.

Embriogénesis, desarrollo larval y sobrevivencia post-asentamiento del coral Orbicella annularis (Scleractinia: Merulinidae)

Introducción: Las poblaciones del coral Orbicella annularis han mostrado bajo reclutamiento en el Caribe. Uno de los cuellos de botella demográficos es la alta mortalidad en las primeras etapas de desarrollo. El conocimiento detallado del ciclo y las tasas de supervivencia de estas fases nos permitirá ayudar en la recuperación de la población y la restauración de los arrecifes. Objetivo: Describir la embriogénesis y estadios larvarios obtenidos por fertilización asistida y medir las tasas de asentamiento y supervivencia de las larvas en sustratos artificiales, antes de ser trasplantadas al arrecife. Métodos: Seis días después de la luna llena de septiembre de 2021, se recolectaron bolsas de gametos de ocho colonias de O. annularis en el Parque Nacional Natural Los Corales del Rosario y San Bernardo, Colombia, y se llevaron al laboratorio. Se realizó fecundación cruzada, se siguió el desarrollo embrionario y larvario hasta el asentamiento larval y se registró supervivencia hasta el día 41. Las larvas se mantuvieron en tres tanques con agua de mar filtrada con 126 sustratos marcados, previamente acondicionados con algas coralináceas costrosas. Luego, los sustratos se trasplantaron al arrecife. Resultados: El inicio del desarrollo embrionario ocurrió 1.11 hAF (horas después de la fertilización), cuando las células mostraron signos de la primera división, y duró hasta 104.59 hAF cuando comenzaron a metamorfosearse. El asentamiento de larvas se observó al sexto día AF. Veintiún días después de la fecundación se encontraron zooxantelas. La supervivencia de las larvas después del asentamiento fue de 27.5 %. Conclusión: En este primer esfuerzo de propagación sexual utilizando O. annularis en Colombia, 1.4 % de larvas competentes completaron todo el proceso de desarrollo. Aunque la tasa de supervivencia fue baja, estos resultados se suman a los esfuerzos de restauración de corales en el Caribe en los que se ayuda a las especies a aumentar la supervivencia de los corales en sus primeras etapas de desarrollo.

Introduction: Populations of the coral Orbicella annularis have shown low recruitment in the Caribbean. One of the demographic bottlenecks is the high mortality in the early stages of development. Detailed knowledge of the cycle and survival rates of these phases will allow us to assist in population recovery and reef restoration. Objective: To describe the embryogenesis and larval stages obtained by assisted fertilization and measure the settlement and survival rates of larvae on artificial substrates, before being outplanted to the reef. Methods: Six days after the full moon in September 2021, gamete bundles were collected from eight O. annularis colonies in Los Corales del Rosario and San Bernardo National Natural Park, Colombia and brought to the laboratory. Cross fertilization was carried out and embryonic and larval development were followed until larval settlement and survival was recorded until day 41. The larvae were kept in three tanks with filtered sea water with 126 tagged substrates, previously conditioned with crustose coralline algae. The substrates were then outplanted to the reef. Results: The onset of embryonic development occurred 1.11 hAF (hours after fertilization), when cells showed signs of the first cleavage, and lasted until 104.59 hAF when they began to metamorphose. Larvae settlement was observed on the sixth day AF. Twenty-one days after fertilization, zooxanthellae were found. Post-settlement larval survival was 27.5 %. Conclusions: In this first sexual propagation effort using O. annularis in Colombia, 1.4 % of competent larvae completed the entire development process. Although low survival rate, these results add to coral restoration efforts in the Caribbean in which species are assisted to increase the survival of corals in their early stages of development.

Single-step genome-wide association studies (GWAS) and post-GWAS analyses to identify genomic regions and candidate genes for milk yield in Brazilian Girolando cattle.

Milk production is economically important to the Brazilian agribusiness, and the majority of the country's milk production derives from Girolando (Gir × Holstein) cows. This study aimed to identify quantitative trait loci (QTL) and candidate genes associated with 305-d milk yield (305MY) in Girolando cattle. In addition, we investigated the SNP-specific variances for Holstein and Gir breeds of origin within the sequence of candidate genes. A single-step genomic BLUP procedure was used to identify QTL associated with 305MY, and the most likely candidate genes were identified through follow-up analyses. Genomic breeding values specific for Holstein and Gir were estimated in the Girolando animals using a model that uses breed-specific partial relationship matrices, which were converted to breed of origin SNP effects. Differences between breed of origin were evaluated by comparing estimated SNP variances between breeds. From 10 genome regions explaining most additive genetic variance for 305MY in Girolando cattle, 7 candidate genes were identified on chromosomes 1, 4, 6, and 26. Within the sequence of these 7 candidate genes, Gir breed of origin SNP alleles showed the highest genetic variance. These results indicated QTL regions that could be further explored in genomic selection panels and which may also help in understanding the gene mechanisms involved in milk production in the Girolando breed.

Genome-wide association studies for heat stress response in Bos taurus × Bos indicus crossbred cattle.

Heat stress is an important issue in the global dairy industry. In tropical areas, an alternative to overcome heat stress is the use of crossbred animals or synthetic breeds, such as the Girolando. In this study, we performed a genome-wide association study (GWAS) and post-GWAS analyses for heat stress in an experimental Gir × Holstein F2 population. Rectal temperature (RT) was measured in heat-stressed F2 animals, and the variation between 2 consecutive RT measurements (ΔRT) was used as the dependent variable. Illumina BovineSNP50v1 BeadChip (Illumina Inc., San Diego, CA) and single-SNP approach were used for GWAS. Post-GWAS analyses were performed by gene ontology terms enrichment and gene-transcription factor (TF) networks, generated from enriched TF. The breed origin of marker alleles in the F2 population was assigned using the breed of origin of alleles (BOA) approach. Heritability and repeatability estimates (± standard error) for ΔRT were 0.13 ± 0.08 and 0.29 ± 0.06, respectively. Association analysis revealed 6 SNP significantly associated with ΔRT. Genes involved with biological processes in response to heat stress effects (LIF, OSM, TXNRD2, and DGCR8) were identified as putative candidate genes. After performing the BOA approach, the 10% of F2 animals with the lowest breeding values for ΔRT were classified as low-ΔRT, and the 10% with the highest breeding values for ΔRT were classified as high-ΔRT. On average, 49.4% of low-ΔRT animals had 2 alleles from the Holstein breed (HH), and 39% had both alleles from the Gir breed (GG). In high-ΔRT animals, the average proportion of animals for HH and GG were 1.4 and 50.2%, respectively. This study allowed the identification of candidate genes for ΔRT in Gir × Holstein crossbred animals. According to the BOA approach, Holstein breed alleles could be associated with better response to heat stress effects, which could be explained by the fact that Holstein animals are more affected by heat stress than Gir animals and thus require a genetic architecture to defend the body from the deleterious effects of heat stress. Future studies can provide further knowledge to uncover the genetic architecture underlying heat stress in crossbred cattle.

Genome-wide association studies for tick resistance in Bos taurus × Bos indicus crossbred cattle: A deeper look into this intricate mechanism.

Rhipicephalus (Boophilus) microplus is the main cattle ectoparasite in tropical areas. Gir × Holstein crossbred cows are well adapted to different production systems in Brazil. In this context, we performed genome-wide association study (GWAS) and post-GWAS analyses for R. microplus resistance in an experimental Gir × Holstein F2 population. Single nucleotide polymorphisms (SNP) identified in GWAS were used to build gene networks and to investigate the breed of origin for its alleles. Tick artificial infestations were performed during the dry and rainy seasons. Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA) and single-step BLUP procedure was used for GWAS. Post-GWAS analyses were performed by gene ontology terms enrichment and gene transcription factors networks, generated from enriched transcription factors, identified from the promoter sequences of selected gene sets. The genetic origin of marker alleles in the F2 population was assigned using the breed of origin of alleles approach. Heritability estimates for tick counts were 0.40 ± 0.11 in the rainy season and 0.54 ± 0.11 in the dry season. The top ten 0.5-Mbp windows with the highest percentage of genetic variance explained by SNP markers were found in chromosomes 10 and 23 for both the dry and rainy seasons. Gene network analyses allowed the identification of genes involved with biological processes relevant to immune system functions (TREM1, TREM2, and CD83). Gene-transcription factors network allowed the identification of genes involved with immune functions (MYO5A, TREML1, and PRSS16). In resistant animals, the average proportion of animals showing significant SNPs with paternal and maternal alleles originated from Gir breed was 44.8% whereas the proportion of animals with both paternal and maternal alleles originated from Holstein breed was 11.3%. Susceptible animals showing both paternal and maternal alleles originated from Holstein breed represented 44.6% on average, whereas both paternal and maternal alleles originated from Gir breed animals represented 9.3%. This study allowed us to identify candidate genes for tick resistance in Gir × Holstein crossbreds in both rainy and dry seasons. According to the origin of alleles analysis, we found that most animals classified as resistant showed 2 alleles from Gir breed, while the susceptible ones showed alleles from Holstein. Based on these results, the identified genes may be thoroughly investigated in additional experiments aiming to validate their effects on tick resistance phenotype in cattle.

Reducing cryptic relatedness in genomic data sets via a central node exclusion algorithm.

Cryptic relatedness is a confounding factor in genetic diversity and genetic association studies. Development of strategies to reduce cryptic relatedness in a sample is a crucial step for downstream genetic analyses. This study uses a node selection algorithm, based on network degrees of centrality, to evaluate its applicability and impact on evaluation of genetic diversity and population stratification. 1,036 Guzerá (Bos indicus) females were genotyped using Illumina Bovine SNP50 v2 BeadChip. Four strategies were compared. The first and second strategies consist on a iterative exclusion of most related individuals based on PLINK kinship coefficient (φij) and VanRaden's φij, respectively. The third and fourth strategies were based on a node selection algorithm. The fourth strategy, Network G matrix, preserved the larger number of individuals with a better diversity and representation from the initial sample. Determining the most probable number of populations was directly affected by the kinship metric. Network G matrix was the better strategy for reducing relatedness due to producing a larger sample, with more distant individuals, a more similar distribution when compared with the full data set in the MDS plots and keeping a better representation of the population structure. Resampling strategies using VanRaden's φij as a relationship metric was better to infer the relationships among individuals. Moreover, the resampling strategies directly impact the genomic inflation values in genomewide association studies. The use of the node selection algorithm also implies better selection of the most central individuals to be removed, providing a more representative sample.

Whole genome sequencing of Guzerá cattle reveals genetic variants in candidate genes for production, disease resistance, and heat tolerance.

In bovines, artificial selection has produced a large number of breeds which differ in production, environmental adaptation, and health characteristics. To investigate the genetic basis of these phenotypical differences, several bovine breeds have been sequenced. Millions of new SNVs were described at every new breed sequenced, suggesting that every breed should be sequenced. Guzerat or Guzerá is an indicine breed resistant to drought and parasites that has been the base for some important breeds such as Brahman. Here, we describe the sequence of the Guzerá genome and the in silico functional analyses of intragenic breed-specific variations. Mate-paired libraries were generated using the ABI SOLiD system. Sequences were mapped to the Bos taurus reference genome (UMD 3.1) and 87% of the reference genome was covered at a 26X. Among the variants identified, 2,676,067 SNVs and 463,158 INDELs were homozygous, not found in any database searched, and may represent true differences between Guzerá and B. taurus. Functional analyses investigated with the NGS-SNP package focused on 1069 new, non-synonymous SNVs, splice-site variants (including acceptor and donor sites, and the conserved regions at both intron borders, referred to here as splice regions) and coding INDELs (NS/SS/I). These NS/SS/I map to 935 genes belonging to cell communication, environmental adaptation, signal transduction, sensory, and immune systems pathways. These pathways have been involved in phenotypes related to health, adaptation to the environment and behavior, and particularly, disease resistance and heat tolerance. Indeed, 105 of these genes are known QTLs for milk, meat and carcass, production, reproduction, and health traits. Therefore, in addition to describing new genetic variants, our approach provided groundwork for unraveling key candidate genes and mutations.

Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

Genomic divergence of zebu and taurine cattle identified through high-density SNP genotyping.

BACKGROUND: Natural selection has molded evolution across all taxa. At an arguable date of around 330,000 years ago there were already at least two different types of cattle that became ancestors of nearly all modern cattle, the Bos taurus taurus more adapted to temperate climates and the tropically adapted Bos taurus indicus. After domestication, human selection exponentially intensified these differences. To better understand the genetic differences between these subspecies and detect genomic regions potentially under divergent selection, animals from the International Bovine HapMap Experiment were genotyped for over 770,000 SNP across the genome and compared using smoothed F(ST). The taurine sample was represented by ten breeds and the contrasting zebu cohort by three breeds. RESULTS: Each cattle group evidenced similar numbers of polymorphic markers well distributed across the genome. Principal components analyses and unsupervised clustering confirmed the well-characterized main division of domestic cattle. The top 1% smoothed F(ST), potentially associated to positive selection, contained 48 genomic regions across 17 chromosomes. Nearly half of the top F(ST) signals (n = 22) were previously detected using a lower density SNP assay. Amongst the strongest signals were the BTA7:~50 Mb and BTA14:~25 Mb; both regions harboring candidate genes and different patterns of linkage disequilibrium that potentially represent intrinsic differences between cattle types. The bottom 1% of the smoothed F(ST) values, potentially associated to balancing selection, included 24 regions across 13 chromosomes. These regions often overlap with copy number variants, including the highly variable region at BTA23:~24 Mb that harbors a large number of MHC genes. Under these regions, 318 unique Ensembl genes are annotated with a significant overrepresentation of immune related pathways. CONCLUSIONS: Genomic regions that are potentially linked to purifying or balancing selection processes in domestic cattle were identified. These regions are of particular interest to understand the natural and human selective pressures to which these subspecies were exposed to and how the genetic background of these populations evolved in response to environmental challenges and human manipulation.

Imputation of microsatellite alleles from dense SNP genotypes for parentage verification across multiple Bos taurus and Bos indicus breeds.

To assist cattle producers transition from microsatellite (MS) to single nucleotide polymorphism (SNP) genotyping for parental verification we previously devised an effective and inexpensive method to impute MS alleles from SNP haplotypes. While the reported method was verified with only a limited data set (N = 479) from Brown Swiss, Guernsey, Holstein, and Jersey cattle, some of the MS-SNP haplotype associations were concordant across these phylogenetically diverse breeds. This implied that some haplotypes predate modern breed formation and remain in strong linkage disequilibrium. To expand the utility of MS allele imputation across breeds, MS and SNP data from more than 8000 animals representing 39 breeds (Bos taurus and B. indicus) were used to predict 9410 SNP haplotypes, incorporating an average of 73 SNPs per haplotype, for which alleles from 12 MS markers could be accurately be imputed. Approximately 25% of the MS-SNP haplotypes were present in multiple breeds (N = 2 to 36 breeds). These shared haplotypes allowed for MS imputation in breeds that were not represented in the reference population with only a small increase in Mendelian inheritance inconsistancies. Our reported reference haplotypes can be used for any cattle breed and the reported methods can be applied to any species to aid the transition from MS to SNP genetic markers. While ~91% of the animals with imputed alleles for 12 MS markers had ≤1 Mendelian inheritance conflicts with their parents' reported MS genotypes, this figure was 96% for our reference animals, indicating potential errors in the reported MS genotypes. The workflow we suggest autocorrects for genotyping errors and rare haplotypes, by MS genotyping animals whose imputed MS alleles fail parentage verification, and then incorporating those animals into the reference dataset.

Novel thalidomide analogues from diamines inhibit pro-inflammatory cytokine production and CD80 expression while enhancing IL-10.

Thalidomide is used to treat a variety of diseases including erythema nodosum leprosum, an inflammatory complication of leprosy. However, this drug has severe teratogenic activity and novel thalidomide analogues might be used to treat diseases without this severe side effect. A series of diamine compounds containing two hydrolyzed phthalimide units were chosen as analogues of thalidomide and evaluated regarding their capacity to regulate the production of molecules involved in inflammatory responses. TNF-α, IL-12 and IL-10 production, and the expression of CD80 and CD86 were investigated in LPS plus IFN-γ-stimulated J774A.1 cells by ELISA and flow cytometry, respectively. The expression of TNF-α and IL-10 mRNA was analyzed by real time RT-PCR. TNF-α, IL-6, IFN-γ, CXCL9 and CXCL10 production by human peripheral blood mononuclear cells (PBMC) were evaluated by flow cytometry. Compounds 3, 6 and 9 greatly inhibited TNF-α and IL-12 production while enhancing IL-10. In addition, CD80 expression was inhibited, but not CD86. The compounds inhibited TNF-α production by PBMC more than thalidomide and also had an inhibitory effect on the production of IL-6, IFN-γ, CXCL9 and CXCL10. Levels of mRNA for TNF-α were reduced after treatment with the compounds, suggesting post- transcriptional effects. The compounds had no effect on cell viability. Our results indicate that the novel diamine compounds 3, 6 and 9 inhibit critical pro-inflammatory cytokines and stimulate IL-10, which make them attractive candidate drugs for the treatment of certain inflammatory conditions and cancer.

Oil production at different stages of leaf development in Lippia alba

The aim of this work was to analyze terpene oil production and terpene synthases (TPS) gene expression from leaves at different developmental stages of different chemotypes of Lippia alba (Mill.) N.E. Br. ex Britton & P. Wilson, Verbenaceae. Hydro-distilled essential oil were used for chemical analysis and gene expression of three monoterpene synthase genes called LaTPS12, LaTPS23 and LaTPS25 were used for analyses of gene expression associated to oil production. The putative genes were associated to TPS-b gene class. Semi-quantitative PCR and quantitative PCR (qPCR) analysis were used to investigate the expression profile of those three putative genes in different leaf stages and different chemotypes. Additionally, total oil production and gene expression of putative TPS genes cloned from L. alba chemotype linalool were evaluated at different stages of leaf development. The expression level of those three genes was higher when the highest oil production was observed, mainly in young leaves at the fourth nodal segment for all evaluated chemotypes. Total oil production was higher at leaves that had unopened trichomes. We also observed that the 1mM of MeJA treatment increased the gene expression in all chemotypes after 24 h elicitation.

Karyotype analysis, DNA content and molecular screening in Lippia alba (Verbenaceae)

Cytogenetic analyses, of pollen viability, nuclear DNA content and RAPD markers were employed to study three chemotypes of Lippia alba (Mill.) (Verbenaceae) in order to understand the genetic variation among them. Different ploidy levels and mixoploid individuals were observed. This work comprises the first report of different chromosome numbers (cytotypes) in L. alba. The chromosome numbers of La2-carvone and La3-linalool chemotypes suggested that they are polyploids. Flow cytometric analysis showed an increase of nuclear DNA content that was not directly proportional to ploidy level variation. A cluster analysis based on RAPD markers revealed that La3-linalool shares genetic markers with La1-citral and La2-carvone. The analysis showed that the majority of genetic variation of La3-linalool could be a consequence of ixoploidy. ur data indicates that sexual reproduction aong those three chemotypes is unlikely and suggests the beginning of reproductive isolation. The results demonstrated that chromosome analysis, nuclear DNA content estimation and RAPD markers constitute excellent tools for detecting genetic variation among L. alba chemotypes.

Análises citogenéticas, de viabilidade do pólen, do conteúdo de DNA nuclear e marcadores RAPD foram empregadas no estudo de três quimiotipos de Lippia alba (Mill.) (Verbenaceae) visando contribuir para o entendimento da variação genética entre os mesmos. Diferentes níveis de ploidia e indivíduos mixoploides foram observados. Este trabalho compreende o primeiro relato de diferentes números cromossômicos (citótipos) em L. alba. Os números cromossômicos dos quimiotipos La2-carvona e La3-linalol sugere que eles seja poliploides. A análise da citometria de fluxo mostrou um aumento do conteúdo de DNA nuclear que não foi diretamente proporcional à variação no nível de ploidia. A análise de agrupamento baseada nos marcadores RAPD demonstrou que La3-linalol compartilha marcadores genéticos com La1-citral e La2-carvona. A análise mostrou que a maior parte da variação genética de La3-linalol pode ser consequência da mixoploidia. Nossos dados indicam que a reprodução sexual entre os três quimiotipos parece improvável, sugerindo o início de isolamento reprodutivo. Os resultados demonstraram que a análise cromossômica, a quantificação do DNA nuclear estimado e os marcadores RAPD constituem excelentes ferramentas para detecção de variação genética entre quimiotipos de L. alba.

Karyotype analysis, DNA content and molecular screening in Lippia alba (Verbenaceae).

Cytogenetic analyses, of pollen viability, nuclear DNA content and RAPD markers were employed to study three chemotypes of Lippia alba (Mill.) (Verbenaceae) in order to understand the genetic variation among them. Different ploidy levels and mixoploid individuals were observed. This work comprises the first report of different chromosome numbers (cytotypes) in L. alba. The chromosome numbers of La2-carvone and La3-linalool chemotypes suggested that they are polyploids. Flow cytometric analysis showed an increase of nuclear DNA content that was not directly proportional to ploidy level variation. A cluster analysis based on RAPD markers revealed that La3-linalool shares genetic markers with La1-citral and La2-carvone. The analysis showed that the majority of genetic variation of La3-linalool could be a consequence of ixoploidy. ur data indicates that sexual reproduction aong those three chemotypes is unlikely and suggests the beginning of reproductive isolation. The results demonstrated that chromosome analysis, nuclear DNA content estimation and RAPD markers constitute excellent tools for detecting genetic variation among L. alba chemotypes.

Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero staphylococcus isoladas de mastite bovina / Identification of staphylococcus strains isolated from bovine mastitis by PCR and 16S rDNA sequencing

O objetivo deste trabalho foi identificar espécies de Staphylococcus (n=100) isoladas de mastite em rebanhos bovinos do Estado de Minas Gerais. Para esta finalidade foram utilizadas reações de PCR empregando oligonucleotídeos iniciadores descritos anteriormente para amplificar genes específicos de S. aureus (femA), S. intermedius (rDNA 16S) e S. hyicus (rDNA 16S-23S) e o sequenciamento do rDNA 16S. De acordo com as reações de PCR, 83 isolados foram identificados como S. aureus, 13 isolados como S. intermedius, dois como S. hyicus e dois isolados não foram identificados. Foram submetidos ao sequenciamento do rDNA 16S seis isolados identificados como S. aureus e os 17 restantes. Os seis isolados identificados como S. aureus confirmaram essa identificação. Dos outros 17 isolados, 13 foram identificados como S. chromogenes e quatro como S. hyicus, com similaridade igual ou superior a 99%. Baseando-se nos resultados da reação de PCR do gene femA e do sequenciamento do rDNA 16S, foram identificados 83 S. aureus, 13 S. chromogenes e quatro S. hyicus. Neste estudo os oligonucleotídeos iniciadores empregados na reação de PCR para S. intermedius não foram específicos, pois amplificaram também S. chromogenes; e os empregados na reação de PCR para S. hyicus não foram sensíveis, pois falharam na identificação de dois isolados de S. hyicus. A identificação definitiva das duas últimas espécies somente foi possível pelo sequenciamento do rDNA 16S.

The objective of this study was to identify the species of 100 isolates of Staphylococcus from mastitis in dairy cows from herds located in the state of Minas Gerais, Brazil. PCR reactions were carried out using specific primers described previously for S. aureus (femA gene), S. intermedius (16S rDNA) and S. hyicus (16S-23S rDNA spacer region). In addition, products of amplification of variable regions of the 16S rDNA gene of the strains were sequenced. According to the results of the PCR, 83 strains were identified as S. aureus, 13 as S. intermedius, two as S. hyicus and two isolates were not identified. The sequencing of 16S rDNA was applied to 23 strains identified by PCR amplifications: six S. aureus and the strains identified as S. intermedius (n=13), S. hyicus (n=2) or not identified (n=2). The sequencing of 16S rDNA confirmed the six strains as S. aureus. The others 17 strains were identified as S. chromogenes (13 isolates) and S. hyicus (four isolates). Each sample was related to a specie according to the smallest E-value and highest similarity (≥ 99%). The identification of S. hyicus and S. chromogenes was accomplished only by 16S rDNA sequencing.

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes.

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O(2); G2, 0.1% polyvinyl alcohol (PVA) with 20% O(2); G3, 10% OCS with 5% O(2); and G4, 0.1% PVA with 5% O(2). The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O(2), whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O(2). The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O(2) (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O(2) provides an efficient in vitro culture condition for the maturation of bovine oocytes.

Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds.

The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.

Cellular and humoral immune responses during intrathoracic paracoccidioidomycosis in BALB/c mice.

Paracoccidioidomycosis is a chronic infection that primarily affects the lungs. Here we investigated cellular and humoral immune responses after intrathoracic Paracoccidioidesbrasiliensis infection in BALB/c mice. P. brasiliensis-colony-forming units (CFUs), fungal DNA and granulomas in lungs increased progressively, peaking at day 90 postinfection (p.i.). IFN-gamma production was highest on day 15 p.i., declining thereafter. The kinetics of the NO production was similar to that described for IFN-gamma. In contrast, IL-10 increased from day 45 p.i. reaching a peak at day 90. Levels of serum IgG1 were higher than IgG2a between days 30 and 90 p.i. 30% of mice died by day 90 p.i. These data indicate that infection with P. brasiliensis by the intrathoracic route shows high IFN-gamma and NO production at day 15 p.i., unable to control multiplication of fungi, which appears to be associated with a progressive increase in IL-10 and in the number and complexity of granulomas.

Translation and validation into the Brazilian Portuguese of the restless legs syndrome rating scale of the International Restless Legs Syndrome Study Group.

BACKGROUND: Restless legs syndrome (RLS) is a chronic sensory-motor disorder characterized by unpleasant limb sensations and an irresistible urge to move. The International Restless Legs Syndrome Study Group developed the Restless Legs Syndrome Rating Scale (IRLS) to assess the severity of RLS symptoms. The objective of this study was to translate and validate the IRLS into Brazilian Portuguese. METHOD: The IRLS was translated into Brazilian Portuguese, analyzed, back translated to English, and compared to the original version. It was applied to 10 patients for cultural verification. The language was adjusted and the final version was administered to 30 patients (13 male, mean age 58.88+/-14.82). RESULTS: There was correlation among the IRLS evaluation of three experts. Many linguistic adaptations were required to achieve cultural adequacy and the Cronbach's alpha coefficient showed reliability of 80%. CONCLUSION: IRLS was translated, adapted, and validated to Brazilian Portuguese language, showing good reliability and validity.