Molecular characteristics and transcriptional regulatory of spermatogenesis-related gene RFX2 in adult Banna mini-pig inbred line (BMI)

RFX2 plays critical roles in mammalian spermatogenesis and cilium maturation. Here, the testes of 12-month-old adult boars of Banna mini-pig inbred line (BMI) were subjected to whole-transcriptome sequencing. The results indicated that the average expression (raw count) of RFX2 gene in BMI testes was 16138.25, and the average expression value of the corresponding transcript ENSSSCT00000043271.2 was 123.1898. The CDS of RFX2 obtained from BMI testes was 2,817 bp (GenBank accession number: OL362242). Gene structure analysis showed that RFX2 was located on chromosome 2 of the pig genome with 19 exons. Protein structure analysis indicated that RFX2 contains 728 amino acids with two conserved domains. Phylogenetic analysis revealed that RFX2 was highly conserved with evolutionary homologies among mammalian species. Other analyses, including PPI networks, KEGG, and GO, indicated that BMI RFX2 had interactions with 43 proteins involving various functions, such as in cell cycle, spermatid development, spermatid differentiation, cilium assembly, and cilium organization, etc. Correlation analysis between these proteins and the transcriptome data implied that RFX2 was significantly associated with FOXJ1, DNAH9, TMEM138, E2F7, and ATR, and particularly showed the highest correlation with ATR, demonstrating the importance of RFX2 and ART in spermatogenesis. Functional annotation implied that RFX2 was involved in 17 GO terms, including three cellular components (CC), six molecular functions (MF), and eight biological processes (BP). The analysis of miRNA-gene targeting indicated that BMI RFX2 was mainly regulated by two miRNAs, among which four lncRNAs and five lncRNAs competitively bound ssc-miR-365-5p and ssc-miR-744 with RFX2, respectively. Further, the dual-luciferase report assay indicated that the ssc-miR-365-5p and ssc-miR-744 significantly reduced luciferase activity of RFX2 3'UTR in the 293T cells, suggesting that these two miRNAs regulated the expression of RFX2. Our results revealed the important role of RFX2 in BMI spermatogenesis, making it an intriguing candidate for follow-up studies.(AU)

Genome-sequence, annotation and phylogenetic insights of the lactic acid bacterium Limosilactobacillus fermentum strain LAB1 obtained from the dairy beverage borhani

Limosilactobacillus fermentum is a promising probiotic with several documented health benefits. LAB1 is an antagonistic L. fermentum strain isolated from borhani, a traditional South Asian beverage prepared from dairy and plant ingredients. Here, I present the genome sequence of the L. fermentum LAB1 strain, its annotation, and phylogenetic features. The 2.01 Mb genome with a G+C content of 51.9% was assembled into 221 contigs and predicted to have 1,913 protein-coding genes, 98 pseudo genes, 7 rRNAs, 60 tRNAs, and 1 CRISPR array. As much as 91.1% of the coding sequences could be assigned to known functional genes. Determination of average nucleotide identity (ANI) of the genome sequence revealed 99.37% identity to that of the type strain ATCC 14931. Its 16S rRNA gene sequence extracted from the genome sequence showed close phylogenetic association with several L. fermentum strains. The genome sequence is expected to provide useful insights with regard to the phenotypic, metabolic and beneficial aspects of this lactic acid bacterium.(AU)

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland

Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.(AU)

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland

Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.(AU)

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Background: Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)

Identification and validation of novel EST-SSR markers in olives

The olive (Olea europaea L.) is a leading oil crop in the Mediterranean area. Limited information on the inheritance of agronomic significant traits hinders progress in olive breeding programs, which encourages the development of markers linked to the traits. In this study, we report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project. Sequences were de novo assembled into 98,924 unigenes, which were then used as a source for microsatellites searching. We identified 923 unigenes that contained 984 SSRs among which dinucleotide SSRs (36 %) were the most abundant, followed by tri- (33 %) and hexa- (21 %) nucleotides. Microsatellite repeat motif GA (37 %) was the most common among dinucleotides, while microsatellite repeat motif GAA was the most abundant trinucleotide SSR motif (16 %). Gene ontology annotations could be assigned to 27 % of the unigenes. A hundred and ten expressed sequence tag-derived-simple sequence repeats (EST-SSRs) with annotated genes were selected for primer designing and finally, 46 (42 %) polymorphic EST-SSRs were successfully amplified and used to validate genetic diversity among 24 olive varieties. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Twenty-seven EST-SSRs showed good diversity properties and were recommended for further olive genome investigation.

Identification and validation of novel EST-SSR markers in olives

The olive (Olea europaea L.) is a leading oil crop in the Mediterranean area. Limited information on the inheritance of agronomic significant traits hinders progress in olive breeding programs, which encourages the development of markers linked to the traits. In this study, we report on the development of 46 olive simple sequence repeat (SSR) markers, obtained from 577,025 expressed sequence tags (ESTs) in developing olive fruits generated in the framework of the Slovenian national olive transcriptome project. Sequences were de novo assembled into 98,924 unigenes, which were then used as a source for microsatellites searching. We identified 923 unigenes that contained 984 SSRs among which dinucleotide SSRs (36 %) were the most abundant, followed by tri- (33 %) and hexa- (21 %) nucleotides. Microsatellite repeat motif GA (37 %) was the most common among dinucleotides, while microsatellite repeat motif GAA was the most abundant trinucleotide SSR motif (16 %). Gene ontology annotations could be assigned to 27 % of the unigenes. A hundred and ten expressed sequence tag-derived-simple sequence repeats (EST-SSRs) with annotated genes were selected for primer designing and finally, 46 (42 %) polymorphic EST-SSRs were successfully amplified and used to validate genetic diversity among 24 olive varieties. The average number of alleles per locus, observed heterozygosity, expected heterozygosity, and polymorphic information content were 4.5, 0.649, 0.604 and 0.539, respectively. Twenty-seven EST-SSRs showed good diversity properties and were recommended for further olive genome investigation.(AU)

Strategies to avoid drowning in the deep sequencing data flood

The enormous technological progress in the field of functional genomics during the last 15 years had a significant impact on animal sciences. With the development of Next Generation Sequencing it became feasible to analyze genomes and transcriptomes within short time frames and affordable costs. One major challenge of this rapid development is to manage the data flood and to perform data analysis and integration in an optimal manner. This review provides some information about a typical analysis pipeline for RNASequencing (RNA-Seq) data and a strategy for the analysis of small RNA-Seq data derived from species with poor annotation for non-coding RNA genes. Furthermore, problems regarding gene annotation in livestock species and their possible implications for data analysis and interpretation are discussed. Despite of not yet solved problems and challenges with respect to data analysis and integration the approaches in the field of functional genome analysis opened up new ways to try to understand the complex trait fertility.

Strategies to avoid drowning in the deep sequencing data flood

The enormous technological progress in the field of functional genomics during the last 15 years had a significant impact on animal sciences. With the development of Next Generation Sequencing it became feasible to analyze genomes and transcriptomes within short time frames and affordable costs. One major challenge of this rapid development is to manage the data flood and to perform data analysis and integration in an optimal manner. This review provides some information about a typical analysis pipeline for RNASequencing (RNA-Seq) data and a strategy for the analysis of small RNA-Seq data derived from species with poor annotation for non-coding RNA genes. Furthermore, problems regarding gene annotation in livestock species and their possible implications for data analysis and interpretation are discussed. Despite of not yet solved problems and challenges with respect to data analysis and integration the approaches in the field of functional genome analysis opened up new ways to try to understand the complex trait fertility.(AU)

GENÔMICA COMPARATIVA DE STAPHYLOCOCCUS SPP. ASSOCIADOS À MASTITE SUBCLÍNICA CAPRINA NA PARAÍBA

Infecções intramamárias acometem frequentemente os animais leiteiros, gerando danos tanto na saúde dos animais e perdas significativas na quantidade e qualidade do leite produzido, como riscos para a saúde dos consumidores tendo em vista que as glândulas mamárias infectadas podem abrigar micro-organismos potencialmente zoonóticos. Os Staphylococcus frequentemente são detectados em exame microbiológico no leite de animais acometidos por infecção mamária. Sendo assim, faz-se necessário estudos mais aprofundados relacionados à resistência antimicrobiana e os fatores de virulência, além de elementos genéticos móveis, afim de minimizar as perdas para a cadeia produtiva e para a saúde pública. O sequenciamento do genoma completo é uma ferramenta desenvolvida para auxiliar com maior facilidade, rapidez e menores custos toda a epidemiologia genômica dos micro-organismos, e, através da anotação funcional, realizar comparações entre micro-organismos e identificar diferenças e similaridades entre elas. O capítulo I, é um referencial teórico, que demonstra aspectos da qualidade do leite de cabras, tanto nos aspectos físico-químicos como microbiológicos, com base na legislação nacional vigente e suas implicações na produção e saúde dos animais e seres humanos. Os capítulos II, III e IV avaliam através do sequenciamento dos genomas, genes de resistência, detecção de plasmídeos, e fatores de virulência de três espécies de Staphylococcus (S.) (S. caprae, S. epidermidis e S. aureus) originários de leite de cabras. As amostras foram submetidas ao teste fenotípico de difusão em discos. Após extração do DNA genômico total e posterior avaliação quali-quantitativa do DNA, foram sequenciadas em tecnologia Illumina Miseq. A montagem de todos os genomas foi executada em Unicycler. A anotação das amostras de S. caprae foi avaliada por meio de análise do pan-genoma. Foi verificada a presença de genes de resistência a tetraciclina, -lactâmicos, fosfomicina, macrolídeos, fenicóis e quinolonas. Já para os fatores de virulência, foram observados aderência, exoenzimas, evasão imune, absorção e metabolism do ferro e toxinas. Os capítulos III e IV, analisam respectivamente as amostras de S. epidermidis e S. aureus a despeito do tipo de sequência multilocus (ST), predição de genes de virulência, plasmídeos rep, tipo de cassete cromossomal e fatores de virulência. Uma amostra de cada uma dessas espécies, detectou o gene mec e tipo de SCCmecIV. S epidermidis exibiu três tipos de sequência multilocus diferentes, resistência genotípica as classes dos macrolídeos, -lactâmicos, tetraciclinas, aminoglicosídeos e fosfomicina e seis diferentes plasmídeos, além de fatores de virulência a aderência, enzimas, evasão imune, toxinas e sistema de secreção. Nas cepas de S. aureus, adicionalmente, foram realizadas análises da proteína A e filogenia. As amostras pertenciam a quatro STs e cinco proteínas A (spa) diferentes. Houve maior prevalência de resistência para os genes de tetraciclina e beta-lactâmicos, entretanto, uma cepa ainda apresentou genes de resistência a trimetropim, macrolídeos, aminogligosídeo e fenicol. Os genes de virulência foram classificados em exoenzimas, toxigênicos e resposta imune. Em resumo, a ocorrência de genes de resistência e plasmídeos portadores de genes de resistência, inclusive, para antimicrobianos de último recurso em infecções humanas mais graves, além de grande frequência de genes de virulência em isolados de Staphylococcus spp. de originários de leite de cabras reforça a preocupação frente à qualidade do leite produzido como alimento seguro.

Intramammary infections often affect dairy animals, causing damage to the health of animals and significant losses in the quantity and quality of milk produced, as well as risks to the health of consumers, given that infected mammary glands can harbor potentially zoonotic microorganisms. Staphylococcus are frequently detected in microbiological examination in the milk of animals affected by mammary infection. Therefore, further studies related to antimicrobial resistance and virulence factors, in addition to mobile genetic elements, are necessary in order to minimize losses to the production chain and public health. The whole genome sequencing is a tool developed to assist with greater ease, speed and lower costs the entire genomic epidemiology of microorganisms, and, through functional annotation, perform comparisons between microorganisms and identify differences and similarities between them. Chapter I is a theoretical framework that demonstrates aspects of goat milk quality, both in physical-chemical and microbiological aspects, based on current national legislation and its implications for the production and health of animals and human beings. Chapters II, III and IV evaluate through genome sequencing, resistance genes, plasmid detection, and virulence factors of three species of Staphylococcus (S.) (S. caprae, S. epidermidis and S. aureus) originating from goats milk. The samples were submitted to the disc diffusion phenotypic test. After extraction of the total genomic DNA and subsequent quali-quantitative evaluation of the DNA, they were sequenced using Illumina Miseq technology. Assembly of all genomes was performed in Unicycler. The annotation of S. caprae samples was evaluated by pan-genome analysis. The presence of resistance genes to tetracycline, -lactams, fosfomycin, macrolides, phenicols and quinolones was verified. As for virulence factors, adherence, exoenzymes, immune evasion, absorption and metabolism of iron and toxins were observed. Chapters III and IV, respectively, analyze S. epidermidis and S. aureus samples regardless of the multilocus sequence type (ST), virulence gene prediction, rep plasmids, chromosomal cassette type and virulence factors. A sample of each of these species detected the mec gene and type of SCCmecIV. S. epidermidis exhibited three different multilocus sequence types, genotypic resistance to macrolides, -lactams, tetracyclines, minoglycosides and fosfomycin and six different plasmids, in addition to adherence virulence factors, enzymes, immune evasion, toxins and secretion system. In the S. aureus strains, additionally, analyzes of protein A and phylogeny were performed. The samples belonged to four different ST's and five different A (spa) proteins. There was a higher prevalence of resistance for the tetracycline and beta-lactam genes, however, one strain still showed resistance genes to trimethoprim, macrolides, aminoglycoside and phenicol. Virulence genes were classified into exoenzymes, toxigenics and immune response. In summary, the occurrence of resistance genes and plasmids carrying resistance genes, including for antimicrobials of last resort in more severe human infections, in addition to the high frequency of virulence genes in Staphylococcus spp. from goats' milk reinforces the concern about the quality of the milk produced as a safe food.

ASSOCIAÇÃO E SELEÇÃO GENÔMICA PARA EFICIÊNCIA ALIMENTAR EM BOVINOS NELORE

Objetivou-se, com este estudo, estimar os parâmetros genéticos para características de eficiência alimentar, crescimento, reprodução e carcaça em rebanhos comerciais de bovinos Nelore, além da resposta correlacionada entre elas. Também objetivou-se realizar um estudo de seleção genômica avaliando métodos de predição, esquemas de validação e pseudo-fenótipos e conduzir um estudo de associação genômica ampla ponderado em passo único e análises de enriquecimento para características relacionadas à eficiência alimentar. Foram avaliados o consumo alimentar residual (CAR), consumo de matéria seca (CMS), conversão alimentar (CA), eficiência alimentar (EA), ganho em peso residual (GPR), consumo e ganho em peso residual (CGR), peso ao nascer (PN), peso aos 120 (P120), 240 (P240), 365 (P365) e 450 (450) dias de idade, perímetro escrotal aos 365 (PE365) e 450 (PE450) dias de idade, área de olho de lombo (AOL), espessura de gordura (EG) e espessura de gordura na garupa (P8). Foram utilizadas informações de crescimento, reprodução e carcaça de 15.639 bovinos Nelore. Foram utilizados dados de testes de eficiência alimentar realizados entre 2011 e 2018, com informações fenotípicas e genotípicas de 4.329 e 3.594 animais, respectivamente. Os parâmetros genéticos foram estimados utilizando método single-step (ssGBLUP). Seis métodos de predição dos valores genômicos (GEBVs) foram utilizados: ssGBLUP, Bayes A, Bayes B, Bayes C, BLASSO e Bayes R. Três esquemas de validação foram utilizados: 1) aleatório: a base de dados foi aleatoriamente dividida em dez subconjuntos e a validação foi realizada em cada subconjunto por vez; 2) idade: a população foi dividida em treinamento e validação com base no ano de nascimento, sendo o primeiro grupo composto por animais nascidos entre 2010 a 2016 e o segundo nascido em 2017; 3) acurácia de predição do valor genético (EBV): foram divididos em dois grupos, sendo os animais com acurácia acima de 0,45 considerados como a população de treinamento; e abaixo de 0,45 a de validação. Foram avaliadas a acurácia e o viés de predição dos GEBVs. A porcentagem da variância explicada por janelas de 10 SNPs consecutivos foram usados para identificar regiões que explicam mais que 0,5% da variância genética aditiva de cada característica. As características relacionadas à eficiência alimentar apresentaram herdabilidades baixas a moderadas, variando de 0,07 a 0,20. As características relacionadas à eficiência alimentar apresentaram correlação genética baixa com crescimento (-0,19 a 0,24), reprodução (-0,24 a 0,27) e carcaça (-0,17 a 0,27), exceto para crescimento com CMS (0,32 a 0,56) e EA (-0,40). Os resultados demonstram que a habilidade de predição foi similares entre os métodos de predição. A baixa herdabilidade obtida, principalmente para EA (0,07±0,03) e CA (0,09±0,03), limitaram as acurácias dos GEBVs que variaram de baixa a moderada. Os coeficientes de regressão das estimativas foram próximos de 1, e similar entre os métodos de predição, esquemas de validação e pseudo-fenótipos. Em média e apesar da baixa variação (0,0331), a validação cruzada aleatória apresentou maior habilidade de predição, variando de 0,07 a 0,037, comparado a acurácia do EBV e idade. A habilidade de predição foi maior para o fenótipo ajustado para efeitos fixos do que para EBV e EBV desregredido (30,0 e 34,3%, respectivamente). As análises de enriquecimento com a ferramenta The Database for Annotation, Visualization and Integrated Discovery (DAVID) revelaram várias vias funcionais como via de sinalização de neuropeptídios (GO: 0007218), regulação negativa da via de sinalização Wnt canônica (GO: 0090090), detecção de estímulos químicos envolvidos na percepção sensorial do sabor amargo (GO: 0001580), atividade do receptor de sabor amargo (GO: 0033038), neuropeptídio atividade hormonal (GO: 0005184), secreção biliar (bta04976), transdução do paladar (bta0742) e via de sinalização de glucagon (bta04922). A seleção para melhorar características de crescimento, reprodução e carcaça pode não alterar CAR, GPR e CGR. Por outro lado, o CMS, EA e CA podem levar a um aumento do peso corporal, além da seleção para CA levar a uma redução no rendimento de carcaça. A natureza genética das características relacionadas à eficiência alimentar pode levar a diferentes respostas genéticas. A escolha do critério de seleção mais adequado depende do sistema e dos objetivos de produção. Os métodos de predição genômica podem fornecer estimativas confiáveis dos valores genômicos para CAR, CMS, GPR e CGR, características que podem ter maior ganho genético e viabilidade de seleção comparada a EA e CA. As análises de enriquecimento mostraram genes associados ao metabolismo de insulina, leptina, glucose, proteína e lipídeo, balanço de energia, estresse oxidativo, sistemas zinc fingers, secreção biliar, saciedade, comportamento alimentar, salivação, digestão e absorção de nutrientes. A identificação das regiões genômicas e seus respectivos genes fornecem informações sobre bases genéticas e regulação biológica da eficiência alimentar em bovinos Nelore.

The aim of this study was to estimate genetic parameters for feed efficiency, growth, reproductive and carcass traits in commercial Nelore cattle herds, and the correlated response between them. It was also aimed perform a study of genomic selection evaluating prediction methods, validation approaches and pseudo-phenotypes, and conduct a weighted single-step genome-wide association study and an enrichment analysis for feed efficiency of feed efficiency related traits. Residual feed intake (RFI), dry matter intake (DMI), feed conversion ratio (FCR), feed efficiency (FE), residual live weight gain (RG), residual intake and live weight gain (RIG), birth weight (BW), weight at 120 (W120), 240 (W240), 365 (W365), and 450 (W450) days of age, scrotal circumference at 365 (SC365) and 450 (SC450) days of age, rib eye area (REA), backfat thickness (BF) and rump fat thickness (RF) were evaluated. The growth, reproductive and carcass traits records from 15,639 Nelore cattle were used. Data from feed efficiency tests carried out between 2011 and 2018, with phenotypic and genotypic information of 4,329 and 3,594 animals, respectively, were considered. The genetic parameters were estimated in a single step approach (ssGBLUP). Six prediction methods of genomic breeding values (GEBVs) were used: ssGBLUP, Bayes A, Bayes B, Bayes C, BLASSO, and Bayes R. Three validation approaches were used: 1) random: the data set was randomly divided into ten subsets and the validation was done in each subset at a time; 2) age: the population was divided into training and validation set based on the year of birth, with the first group consisting of animals born between 2010 and 2016 and the second group born in 2017; 3) genetic breeding value (EBV) accuracy: were divided into two groups, with animals with accuracy above 0.45 considered as the training population, and below 0.45 the validation set. We checked the accuracy and bias of GEBV. The percentage of variance explained by windows of 10 adjacent SNPs was used to identify regions that explained more than 0.5% of the additive genetic variance on each trait. The feed efficiency related traits showed low to moderate heritabilities, ranging from 0.07 to 0.20. Feed efficiency related traits showed low genetic correlations with growth (-0.19 to 0.24), reproductive (-0.24 to 0.27) and carcass (-0.17 to 0.27) traits, except for growth with DMI (0.32 to 0.56) and FE (-0.40). The results showed that the prediction ability were similar between the prediction methods. The low heritability obtained, mainly for FE (0.07±0.03) and FCR (0.09±0.03), limited the GEBVs accuracy, which ranged from low to moderate. The regression coefficient estimates were close to 1, and similar between the prediction methods, validation approaches, and pseudo-phenotypes. On average and despite low variation (0.0331), the random cross-validation presented the most accurate predictions, ranging from 0.07 to 0.037, than EBV accuracy and age. The prediction ability was higher for phenotype adjusted for fixed effects than for EBV and EBV deregressed (30.0 and 34.3%, respectively). Enrichment analysis by The Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed several functional vias such as neuropeptide signaling pathway (GO:0007218), negative regulation of canonical Wnt signaling pathway (GO:0090090), detection of chemical stimulus involved in sensory perception of bitter taste (GO:0001580), bitter taste receptor activity (GO:0033038), neuropeptide hormone activity (GO:0005184), bile secretion (bta04976), taste transduction (bta0742), and glucagon signaling pathway (bta04922). The selection to improve growth, reproductive and carcass traits would not change RFI, RG, and RIG. On the other hand, DMI, FE and FCR may lead to an increase in body weight, in addition to the selection for FCR may lead to a reduction in carcass yield. The genetic background of feed efficiency related traits are different, which would lead to different genetic responses. The choice of the most adequate selection criterion depends on the production system and goals. Genomic prediction methods can provide a reliable estimate of genomic breeding values for RFI, DMI, RG and RGI, traits that may have higher genetic gain and selection viability than FE and FCR. Enrichment analyzes showed genes associated with in insulin, leptin, glucose, protein and lipid metabolism, energy balance, heat and oxidative stress, zinc finger system, bile secretion, satiety, feed behavior, salivation, digestion and absorption of nutrients. The identification of these genomic regions and their respective genes provide information about genetic basis and biologic regulation for Nelore feed efficiency related traits.

IDENTIFICATION OF STRUCTURAL VARIANTS AND SELECTION SIGNATURES IN CATTLE

Devido aos impactos causados na produção animal recorrentes das mudanças climáticas, é importante caracterizar o genoma bovino para desvendar os mecanismos genéticos envolvidos na variação fenotípica que foram influenciados pelo ambiente e moldados pela seleção natural. O objetivo deste estudo é descrever os principais efeitos da adaptação e seleção em animais zebuínos e taurinos localmente adaptadas através da identificação de variações estruturais e assinaturas de seleção utilizando dados genotípicos e de sequenciamento de genoma inteiro. No capítulo 2, foram utilizados genótipos imputados (n=735.044 marcadores) de 9,386 animais da raça Nellore e de suas respectivas linhagens a fim de estimar a autozigosidade do genoma baseado nas corridas de homozigose (ROH) por meio do software Plink. Em geral, os coeficientes de endogamia baseados em ROH (FROH) não foram altos, com valores próximos a 2%. As ilhas de autozigosidade foram evidentes em todo o genoma e sua localização não diferiu em grande número dentro das linhagens. Termos enriquecidos (p<0,01) dentro das ilhas de autozigosidade sugeriam uma forte seleção para características relacionadas à resposta imune, podendo explicar uma maior adaptabilidade do gado zebuíno em ambientes severos. O capítulo 3 visou avaliar a autozigosidade de todo o genoma para explorar regiões ricas em ROH que poderiam melhor caracterizar os diferentes tipos biológicos (produtivo ou adaptativo) do gado de corte composto Montana Tropical®. Animais Montana (n=1.436) foram genotipados com o GGP-LD BeadChip (n=30.105 marcadores) e os ROH foram identificados em cada indivíduo usando o software Plink. O número de ilhas de autozigosidade não diferiu consideravelmente entre os tipos biológicos e não foi encontrado nenhum termo enriquecido significativo (p<0,05) compartilhado entre eles. Termos enriquecidos associados à resposta imunológica e homeostase foram descritos para o tipo biológico adaptativo, enquanto aqueles ligados ao sistema imunológico, bem como às funções reprodutivas e produtivas, foram identificados para o tipo biológico produtivo. No capítulo 4, quatro métodos estatísticos foram implementados para detectar regiões genômicas sob pressão seletiva usando dados de sequenciamento de genoma inteiro (~12.4 X) de bovinos das raças Gir (GIR, n=13), Caracu Caldeano (CAR, n=12), Crioulo Lageano (CRL, n=12) e Pantaneiro (PAN, n=12). As estatísticas dentro de população (CLR e iHS) e entre populações (FST e XPEHH) foram combinadas separadamente em um único valor por meio do método de-correlated composite of multiple signals (DCMS). As regiões de varredura seletiva foram identificadas por meio dos valores do limite superior (1%) da distribuição empírica gerada por cada estatística DCMS. As assinaturas de seleção identificadas forneceram uma percepção abrangente de genes candidatos juntamente com QTLs relacionadas a características produtivas e de adaptação ao ambiente hostil no qual estas raças foram expostas. No capítulo 5, o método de leitura baseada em read-depth implementado no software CNVnator foi utilizado para identificar variações no número de cópias (CNVs) utilizando dados de sequenciamento de genoma inteiro (~14.07 X) de bovinos das raças CAR (n=12), CRL (n=12) e PAN (n=12). Regiões de CNV (CNVRs) foram identificadas sobrepondo as CNVs individuais dentro de cada raça. A anotação funcional das CNVRs revelou variantes com elevada consequência na sequência proteica abrangendo genes fortemente associados a resiliência ambiental, dentre os quais podemos destacar o BOLA-DQB, BOLA-DQA5, CD1A, -defensins, PRG3 e ULBP21. A análise de enriquecimento funcional utilizando os genes prospectados nas CNVRs também revelou termos significativos (p<0.01) fortemente associados à imunidade e resistência do gado a ambientes severos. Nossos resultados elucidaram os mecanismos biológicos inerentes as raças bovinas aqui estudadas, fornecendo informações a respeito de genes candidatos e regiões genômicas que abrangem características adaptativas relevantes, bem como informações úteis para futuras abordagens de conservação, estudos de associação ou seleção.

Given the impacts caused by climate change upon livestock production, it is important to characterize the cattle genome to unravel the genetic mechanisms underlying phenotypic variation that were influenced by the environment and shaped by natural selection that allowed them to thrive in distinct ecosystems. Therefore, the objective of this study is to describe the main effects of adaptation and selection in indicine and locally adapted taurine cattle breeds through the identification of structural variants and signatures of selection using genotypic and whole-genome re-sequencing data. In chapter 2, imputed genotypes (n=735,044 markers) were used to assess genome-wide autozygosity based on runs of homozygosity (ROH) in 9,386 Nellore animals and its lineages using the Plink software. Overall, inbreeding coefficients based on ROH (FROH) were not high, with values close to 2%. Autozygosity islands were evident across the genome, and their genomic location did not largely differ within lineages. Enriched terms (p<0.01) within the autozygosity islands suggested a strong selection for immune response-related traits and might explain the greater adaptability of the indicine cattle in harsh environments. Chapter 3 aimed to assess genome-wide autozygosity to explore ROH hotspot regions which could better characterize the different biological types (productive or adaptive) within the composite Montana Tropical® beef cattle. Montana animals (n=1,436) were genotyped with the GGP-LD BeadChip (n=30,105 markers), and ROH were identified in every individual using the Plink software. The number of autozygosity islands did not differ considerably between biological types, and no significant enriched term (p<0.05) was found to be shared between them. Enriched terms associated with the immune response and homeostasis were described for the adaptive biological type, while those linked to the immune system as well as with reproductive and productive functions we identified for the productive biological type. In chapter 4, four statistical methods were implemented to detect genomic regions under selective pressure using whole-genome re-sequencing data from Gir (GIR, n=13), Caracu Caldeano (CAR, n=12), Crioulo Lageano (CRL, n=12), and Pantaneiro (PAN, n=12) cattle breeds. Within-population (CLR and iHS) and cross-population statistics (FST and XPEHH) were combined separately in a single score using the de-correlated composite of multiple signals (DCMS) method, and putative sweep regions were revealed by assessing the top 1% of the empirical distribution generated by each DCMS statistic. The signatures of selection identified herein provided a comprehensive set of putative candidate genes together with QTLs disclosing cattle production traits and adaptation to the challenging environment in which these breeds have been exposed. In chapter 5, the read depth-based method implemented in CNVnator was used for copy number variants (CNV) calling on re sequenced data (~14.07 X) from CAR (n=12), CRL (n=12), and PAN (n=12) cattle breeds. CNV regions (CNVRs) were identified by overlapping individual CNVs within each breed. The functional annotation of the CNVRs revealed variants with high consequence on protein sequence harboring relevant genes with functions strongly linked to environmental resilience (i.e., BOLA-DQB, BOLA-DQA5, CD1A, -defensins, PRG3, and ULBP21). Enrichment analysis based on the gene list retrieved from the CNVRs also disclosed over-represented terms (p<0.01) greatly associated with immunity and cattle resistance to harsh environments. Our findings improve the knowledge about the genome biology of such cattle breeds and provide candidate genes and genomic regions encompassing relevant traits as well as useful information for future conservation, association, or selection approaches.

IDENTIFICATION AND FUNCTIONAL ANNOTATION OF GENES RELATED TO HORSES PERFORMANCE: FROM GWAS TO POST-GWAS

O desempenho atlético de cavalos é uma característica influenciada por um grande número de genes. Porém, essa característica tem sido pouco estudada no âmbito da genética molecular, havendo poucos genes candidatos identificados para o desempenho atlético em cavalos. Objetivou-se com este estudo: 1 / identificar genes candidatos associados a adaptações fisiológicas no condicionamento físico de cavalos através de uma revisão sistemática e 2 / construir redes de genes a partir dos genes candidatos identificados, com o objetivo de destacar os genes mais candidatos ao desempenho de cavalos. Uma revisão sistemática realizada por dois avaliadores independentes buscou artigos revisados por pares usando 20 combinações de palavras-chave. A partir disso, nove artigos foram selecionados e definidos como grupos para análise funcional. Um total de 669 genes candidatos foram identificados e usados para análises adicionais. Redes de gene-processos biológicos de cada grupo de genes foram construídas destacando processos associados ao desempenho de cavalos(por exemplo, regulação da pressão arterial sistêmica por vasopressina; regulação da polimerização e despolimerização de actina; secreção de glicocorticoides). Além disso, fatores de transcrição associados a genes candidatos foram identificados. Com base nos processos biológicos(por exemplo, regulação do fator de crescimento endotelial vascular e desenvolvimento do sistema esquelético) e evidências da revisão da literatura, conseguimos identificar os principais FTs relacionados ao desempenho de cavalos em cada grupo, o que nos permitiu construir uma rede gene-FT destacando o FTe a maioria dos genes candidatos ao desempenho de cavalos. A partir da rede gene-FT, pudemos observar quatro FTs principais (TFAP2A, ARNT, EGR1eSP1) e53 genes candidatos (por exemplo, PPP4R2, PDLRN3, IFNAR1e LOC100071438) relacionados ao desempenho em equinos.

The athletic performance of horses is a trait influenced by a large number of genes. However, this trait has been little studied in the field of molecular genetics, with few candidate genes identified for athletic performance in horses. The objective of this study was to: 1/identify candidate genes associated with physiological adaptations in the physical conditioning of horses via systematic review, and 2/build gene networks from the identified candidate genes aiming to highlight the most candidate genes for horse performance. A systematic review performed by two independent judges search for peer-reviewed articles using 20 combinations of keywords. From that, nine articles were selected and defined as a group for functional analysis. A total of 669candidate genes identified and used for further analyses. Gene networks of biological processes from each group set of genes were constructed highlighting processes associated with horse performance (e.g. regulation of systemic arterial blood pressure by vasopressin; regulation of actin polymerization and depolymerization; and glucocorticoid secretion). Besides, transcription factors associated with candidate genes were identified. Based on biological process (e.g. Regulation of vascular endothelial growth factor and Skeletal system development) and evidences from literature review, we were able to identify the main TFs related to horse performance in each group, which allowed us to construct a gene-TF network highlighting the TF and most candidate genes for horse performance. From the gene-TF network we were able to observe four main TFs (TFAP2A, ARNT, EGR1, and SP1) and 53most candidate genes (e.g. PPP4R2, PDLRN3, IFNAR1, and LOC100071438) related to horse performance.

Análise funcional de genes candidatos para características de interesse econômico em bovinos da raça Canchim

Sabe-se que a qualidade de um produto é o fator fundamental para que o mesmo possa ser inserido no mercado e o setor brasileiro de carne bovina tem se preocupado não só com o aumento da produção, mas também com a qualidade da carne produzida. No entanto, produtores enfretam dificuldades para obter a melhoria de características como a quantidade de gordura subcutânea e entremeada, ganho de peso, além da pelagem e da circunferência escrotal. Diversas metodologias foram desenvolvidas no intuito de obter resultados mais rápidos e mais acurados, como o uso de genes candidatos e análises individuais e adjacentes de SNPs que controlam a manifestação de características de interesse econômico, assim como estudos que visam elucidar a arquitetura biológica e metabólica envolvida na característica. Diante do exposto, o presente estudo tem como objetivo realizar uma análise funcional de genes obtidos de regiões genômicas previamente idenficadas por estudos de associação ampla do genoma (GWAS). Foram prospectadas pelo banco de dados Ensembl, 36 regiões genômicas associadas com com espessura de gordura subcutânea (EGS), marmoreio (MARM), área de olho de lombo (AOL), peso ao sobreano (PESO), pelagem (PELO) e circunferência escrotal (CE). Os 220 genes encontrados foram submetidos à anotação funcional pela ferramenta Database for Annotation, Visualization, and Integrated Discovery (DAVID) para verificar suas funcionalidades nas categorias processo biológico, componente celular, função molecular e vias metabólicas, e foram obtidas 186 anotações. Para a verificação das vias metabólicas, foi utilizado o banco de dados Kyoto Encyclopedia of Genes and Genomes (KEGG). Posteriormente, foram selecionados os genes candidatos a partir da filtragem de termos ontológicos e de vias metabólicas relacionados com tecido adiposo, tecido muscular, desenvolvimento e proliferação celular, pelagem e bolsa escrotal, sendo estes obtidos pelos bancos de dados QuickGO e Integrative Visual Analysis Tool for Biological Networks and Pathways (VisANT). Outro critério de seleção utilizado para a seleção de genes foi a identificação prévia dos mesmos na literatura. Após esta filtragem, foram selecionados 29 genes considerados como potenciais candidatos para as características avaliadas, sendo dois genes para EGS, três para MARM, 15 para AOL, seis para PESO, um para PELO e cinco para CE. Os resultados obtidos neste estudo podem na seleção de animais geneticamente superiores em programas de melhoramento da raça Canchim.

It is known that the quality of a product is the fundamental factor so that it can be inserted in the market and the Brazilian beef sector has been concerned not only with the increase of production, but also with the quality of the meat produced. However, producers face some difficulties to obtain improvement of characteristics such as the amount of back fat and marbling, weight gain, besides the coat and the scrotal circumference. Several methodologies have been developed in order to obtain faster and more accurate results, such as the use of candidate genes and individual and adjacent analyzes of SNPs that control the manifestation of characteristics of economic interest, as well as studies aimed at elucidating the biological and metabolic architecture involved in the feature. In view of the above, the present study aims to perform a functional analysis of genes obtained from genomic regions previously identified by genome-wide association study (GWAS). A total of 36 genomic regions has been associated with back fat thickness (BFT), marbling (MARB), rib eye area (REA), yerling weight (YW), coat (COAT) and scrotal circumference (SC). The 220 genes were submitted to the functional annotation by the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool to verify their functionalities in the categories biological process, cellular component, molecular function and metabolic pathways, and 186 annotations were obtained. For the verification of the metabolic pathways, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used. Later, the candidate genes were selected from the filtering of ontological terms and metabolic pathways related to adipose tissue, muscle tissue, cell development and proliferation, coat and scrotal pouch, obtained by the QuickGO and Integrative Visual Analysis Tool for Biological Networks and Pathways (Visant). Another selection criteria used for the selection of genes was the previous identification of the same ones in the literature. After this filtering, 29 genes were considered as potential candidates for the characteristics evaluated, beeing two genes for BFT, three for MARB, 15 for REA, six for YW, one for COAT and five for SC. The results obtained in this study can be helpfull for the selection of genetically superior animals in breeding programs of the Charbray breed.

Transcriptoma comparativo e redes de co-expressão gênica associados a características de carcaça de bovinos Nelore

O objetivo deste trabalho foi estudar o transcriptoma do músculo Longissimus dorsi (LD) de bovinos da raça Nelore associado as características de área de olho de lombo (AOL) e espessura de gordura subcutânea (EGS), a fim de encontrar genes diferencialmente expressos (GDE), conjuntos de genes co-expressos, vias metabólicas e processos biológicos que regulam essas características. Foram utilizados dados de 385 bovinos Nelore castrados para obtenção das medidas fenotípicas de AOL e EGS, mensuradas entre a 12ª e 13ª costelas do músculo LD. Estes animais foram separados em dois grupos extremos, alto e baixo, contendo seis animais cada, baseados nos valores genômicos estimados (GEBV), para AOL e para EGS. Estes conjuntos de 12 animais foram submetidos à análise de expressão diferencial afim de encontrar GDE entre os grupos. Em seguida, a análise de enriquecimento funcional a partir da lista de GDE foi executada por meio do DAVID (Database for Annotation, Visualization and Integrated Discovery) e da combinação do BiNGO (Biological Networks Gene Ontology) e REVIGO (Reduce + Visualise Gene Ontology). Para AOL, foram identificados 101 GDE entre os grupos de alto e baixo GEBV. Desses, 72 genes apresentaram-se down-regulated e 29 up-regulated no grupo baixo. Para EGS, foram encontrados 18 GDE, dos quais treze apresentaramse up-regulated e cinco down-regulated no grupo baixo. Dentre as vias metabólicas e processos biológicos encontrados para essas características, destacam-se a via de sinalização MAPK (bta04010) e a via da endocitose (bta04144) AOL e, os processos de biossíntese de andrógenos (GO:0006702) e via de sinalização canônica Wnt (GO:0060070) EGS. Para encontrar conjuntos de genes co-expressos associados aos tratamentos (AOL e EGS), foi utilizado o pacote WGCNA (Weighted Correlation Network Analysis) do R, com dados de contagens de transcritos de 43 animais. Foram identificados 37 módulos, dentre eles, os módulos Azul, Verde-escuro e Salmon apresentaram correlação significativa de 0,3 com a EGS (P<0,10). Os genes destes módulos foram selecionados para análise de enriquecimento funcional quando seus valores de filiação ao módulo foram superiores a 0,7. O módulo azul apresentou o maior número de genes co-expressos, com 953 genes submetidos à análise de enriquecimento funcional. Foram encontradas seis vias metabólicas e 101 termos do Gene Ontology para este módulo, conforme análise do DAVID, além de 108 processos biológicos identificados pelo BiNGO/REVIGO. Os resultados do presente estudo enfatizam a complexidade da regulação gênica do músculo LD de bovinos Nelore associado às características de AOL e EGS. Estes resultados nos auxiliam a compreender melhor os diversos processos moleculares envolvidos na deposição muscular e de gordura de cobertura, características de carcaça economicamente importantes para a produção da carne bovina.

The aim of this work was to study the Longissimus dorsi (LD) muscle transcriptome of Nellore cattle associated with ribeye area (REA) and backfat thickness (BFT) to find differentially expressed genes (DEG), coexpressed gene modules, metabolic pathways, and biological processes that regulate these traits. Data from 385 Nellore steers were used to obtain the phenotypic measures of REA and BFT, measured between the 12th and 13th ribs of the LD muscle. These animals were divided into two extreme groups, high and low, with six animals each, based on the estimated genomic breeding values (GEBV) for REA and for BFT. These sets of 12 animals were submitted to differentially expressed gene analysis to find DEG between the groups. Then, the functional enrichment analysis from the list of DEG was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery) and the combination of BiNGO (Biological Networks Gene Ontology) and REVIGO (Reduce + Visualize Gene Ontology). For REA, 101 DEG were identified between the high and low GEBV groups. Of those, 72 genes were down-regulated and 29 up-regulated in the low group. For BFT, 18 DEG were found, of which thirteen were up-regulated and five were downregulated in the low group. Among the metabolic pathways and biological processes found for these traits, the MAPK signaling pathway (bta04010) and the endocytosis pathway (bta04144) for REA , the androgen biosynthetic processes (GO: 0006702) and the canonical Wnt signaling pathway (GO: 0060070) for EGS can be highlighted. To find coexpressed gene modules associated with the traits (REA and BFT), we used the WGCNA (Weighted Correlation Network Analysis) package from R, with data from transcript counts of 43 animals. A total of 37 modules were founded. Among them, the Blue, Dark Green and Salmon modules had a significant correlation of 0.3 with BFT (P<0.10). The genes of these modules were selected for functional enrichment analysis when their module membership values were higher than 0.7. The blue module had the highest number of co-expressed genes, with 953 genes submitted to functional enrichment analysis. Six metabolic pathways and 101 Gene Oontology terms were founded for this module by DAVID analysis, in addition, 108 biological processes were identified by BiNGO/REVIGO. The results of the present study emphasize the complexity of gene regulation in the LD muscle of Nellore cattle associated with REA and BFT. These results can help us to better understand the different molecular processes involved in muscle and fat deposition, which are economically important carcass traits for beef production.

Integrative analysis of microRNAs and mRNAs involved in regulation of intramuscular fat deposition in Nelore cattle

A quantidade de gordura intramuscular pode influenciar as características sensoriais e o valor nutricional da carne bovina, assim, a seleção de animais com conteúdo de gordura adequado para o consumidor torna-se importante. A gordura intramuscular é uma característica complexa, de difícil medição e há um conhecimento crescente sobre os genes e vias que controlam os processos biológicos envolvidos na deposição de gordura no músculo. MicroRNAs (miRNAs) são uma classe bem conservados de pequenos RNAs não-codificantes, que modulam a expressão gênica de uma gama de funções no desenvolvimento e fisiologia animal. Este estudo objetivou identificar miRNAs diferencialmente expressos (DE), genes reguladores candidatos e redes de co-expressão usando dados de expressão de mRNAs e miRNAs do músculo Longissimus dorsi de 30 novilhos Nelore com valores genéticos genômicos estimados (GEBV) extremos para conteúdo de gordura intramuscular (IMF). A análise de expressão diferencial entre os dados de miRNA de animais com valores extremos de GEBV para o IMF identificou seis miRNAs DE. A anotação funcional de genes alvos destes microRNAs indica que a via de sinalização de PPAR está envolvida com a deposição de IMF. Os genes reguladores candidatos, tais como SDHAF4, FBXO17, ALDOA e PKM foram identificados pelas abordagens de correlação parcial com teoria da informação (PCIT), fator de impacto fenotípico (PIF) e fator de impacto regulatório (RIF) a partir de dados integrados de expressão de mRNAs-miRNAs. Dois miRNAs, bta-miR-143 e bta-miR-146b, com alta expressão no grupo de baixo conteúdo de IMF, também foram correlacionados com genes reguladores candidatos, os quais foram funcionalmente enriquecidos para termos GO relacionados a oxidação de ácidos graxos. As redes de co-expressão identificaram vários módulos relacionados ao sistema imunológico, ao metabolismo das proteínas, ao metabolismo energético e ao catabolismo da glicose através da análise ponderada da rede de correlação (WGCNA), que mostrou possível interação e regulação entre mRNAs e miRNAs. Este estudo contribui com a compreensão dos possíveis mecanismos reguladores das redes de sinalização genética envolvidas no processo de deposição de gordura. O metabolismo da glicose e o processo de inflamação foram as principais vias encontrados na análise integrada de mRNA-miRNA e mostraram estar associadas ao conteúdo de gordura intramuscular em bovinos de corte.

The amount of intramuscular fat can influence the sensory characteristics and nutritional value of beef, thus the selection of animals with adequate fat content for consumer becomes important. Intramuscular fat is a complex trait that is difficult to measure and there is growing knowledge about the genes and pathways that control the biological processes involved in fat deposition in muscle. MicroRNAs (miRNAs) are well conserved class of non-coding small RNAs that modulate gene expression of a range of functions in animal development and physiology. This study aimed to identify differentially expressed (DE) miRNAs, regulatory candidate genes and co-expression networks using mRNAs and miRNAs expression data from the Longissimus dorsi muscle of 30 Nelore steers with extreme genomic estimated breeding values (GEBV) for intramuscular fat (IMF) content. The differential expression analysis between the miRNA data from animals with extreme GEBV values for IMF identified six DE miRNAs. Functional annotation of target genes for these microRNAs indicates that PPARs signaling pathway is involved with IMF deposition. Regulatory candidate genes such as SDHAF4, FBXO17, ALDOA and PKM were identified by partial correlation with information theory (PCIT), phenotypic impact factor (PIF) and regulatory impact factor (RIF) approaches from integrated miRNAs-mRNAs expression data. Two DE miRNAs, bta-miR-143 and bta-miR-146b, upregulated in Low IMF group, were also correlated with regulatory candidate genes, which were functionally enriched for GO terms for fatty acids oxidation. Co-expression networks identified several modules related to immune system, protein metabolism, energy metabolism and glucose catabolism by weighted correlation network analysis (WGCNA), which showed possible interaction and regulation between mRNAs and miRNAs. This study contributes to our understanding of regulatory mechanisms of gene signaling networks involved in fat deposition process. Glucose metabolism and inflammation process were the main pathways found in integrative mRNAs-miRNAs analysis and showed to influence intramuscular fat content in beef cattle.

PROSPECÇÃO DE NOVOS ALVOS TERAPÊUTICOS EM Corynebacterium pseudotuberculosis A PARTIR DE DADOS DO GENOMA CENTRAL.

Corynebacterium pseudotuberculosis é um microrganismo Gram-positivo, pleomórfico, intracelular facultativo e agente etiológico de doenças de relevância veterinária, como linfadenite caseosa e linfangite ulcerativa, que afetam diferentes espécies animais e causam danos ao agronegócio mundial. Os fatores de virulência desta bactéria são bem caracterizados, como fosfolipase D e ácidos micólicos. Entretanto, ainda não existem métodos de tratamento efetivos para contornar os impactos causados pelo patógeno. Devido a importância deste organismo, muitas linhagens já foram sequenciadas, e o aumento de genomas depositados em bancos de dados públicos permitiu a execução de estudos relacionados à genômica comparativa, em especial, o pan-genoma, que é constituído de três partes: genoma central, genoma acessório e genes linhagem-específicos. O genoma central consiste em genes que são compartilhados por todas as linhagens analisadas, e eles geralmente codificam proteínas que são essenciais para mecanismos de manutenção das atividades celulares, e esta característica faz deles interessantes objetos de estudo para a busca de novos alvos terapêuticos. Além disto, estudos de transcriptômica foram realizados para analisar a expressão de genes deste patógeno sob as condições de estresse ácido (pH 5), osmótico (2M) e térmico (50ºC). O objetivo deste estudo foi obter o genoma central de C. pseudotuberculosis e analisar o perfil transcriptômico diferencial nas três condições de estresse das linhagens 1002 (biovar ovis) e 258 (biovar equi), disponíveis em bancos de dados públicos para prospectar novos alvos terapêuticos. O programa PGAP foi usado para analisar o genoma de 57 linhagens depositadas no NCBI. Os genes diferencialmente expressos foram identificados pelos protocolos dos programas TopHat e Cufflinks, considerando umas expressão mínima de duas vezes (fold change 2) em relação à condição controle. A anotação funcional do genoma central foi executada pela ferramenta GO feat. Os candidatos que apresentaram expressão diferencial foram selecionados para análises modelagem molecular através do pacote MHOLline. O pan-genoma da espécie possui 6243 genes, os quais 897 (14,3%) pertencem ao genoma central. Foram identificados sete genes alvo diferencialmente expressos (panC, tlyC, dps, secE, hemK, bioY e bioM) com funções relacionadas à virulência da bactéria. Destes, o gene dps, que codifica uma proteína relacionada à proteção do DNA perante estresses obteve elevados índices de expressão (fold change = 6,16 e 7,63) na condição ácida e bons resultados de modelagem, com 55.4% de identidade com a estrutura-molde, como um excelente alvo terapêutico em C. pseudotuberculosis.

Corynebacterium pseudotuberculosis is a Gram-positive microorganism, pleomorphic, facultative intracellular and etiologic agent of veterinary relevance diseases, such as caseous lymphadenitis and ulcerative lymphangitis, affecting different animal species which causes damage to the global agribusiness. The virulence factors of this bacterium are well characterized, like phospholipase D and mycolic acids. However, there are still no completely effective treatment methods to overcome the impacts of this pathogen. Due to the importance of this organism, several strains have been sequenced, and the increase of genomes deposited on public databases allowed the execution of studies related to comparative genomics, specially, the pan-genomic approach, that is composed of three parts: core genome, accessory genome and strain-specific genes. The core genome consists in genes that are shared by all strains analyzed, and they usually encode proteins that are essential to mechanisms of maintenance of cellular activities, and this characteristic makes them interesting objects of studies for new therapeutic targets. Furthermore, transcriptomic studies were done to analyze the gene expression of this pathogen under acid (pH 5), osmotic (2M) and thermal (50ºC) stress conditions. The aim of this study was obtain the core genome of C. pseudotuberculosis and analyze the differential transcriptomic profile in three stress conditions of strains 1002 (biovar ovis) and 258 (biovar equi), available on public databases, to prospect new therapeutic targets. PGAP software was used to analyze the pan-genome of 57 strains available on NCBI. The differentially expressed genes were identified by the protocols of Tophat and Cufflinks, considering a minimum expression alteration of two fold (fold change 2) in relation of control condition. The functional annotation of core genome was performed by GO feat. Candidates who presented differential expression were selected for analysis of molecular modeling in the MHOLline software. The pan-genome of the species has 6243 genes, of which 897 (14.3%) belong to the central genome. Were identified seven target genes differentially expressed (panC, tlyC, dps, secE, hemK, bioY and bioM) with functions related to the virulence of the bacterium. Among them, the dps gene, which encodes a protein related to DNA protection under stress, had high expression rates (fold change = 6.16 and 7.63) in the acid condition and 55.4% identity with the protein template, as an excellent therapeutic target in C. pseudotuberculosis.

EXPRESSÃO GÊNICA DIFERENCIAL EM ÚBERE EXTRACORPÓREO DE VACAS MESTIÇAS HOLANDÊS-ZEBU INFECTADO POR Streptococcus agalactiae

A mastite é responsável por grandes perdas econômicas na bovinocultura leiteira e necessita de maior compreensão de suas bases genéticas para que métodos de melhoramento genético possam ser desenvolvidos. Neste sentido, estudos de expressão gênica têm sido empregados. Portanto, os objetivos deste trabalho foram: 1. Detectar, por meio de três métodos computacionais distintos, a expressão gênica diferencial de dados obtidos por sequenciamento de RNA extraído de glândula mamária mantida em um sistema extracorpóreo de bovinos de leite mestiços Holandês-Zebu e infectada experimentalmente com Streptococcus agalactiae; 2. Estudar o perfil de expressão do total de genes identificados e aqueles comuns aos três métodos relacionados ao desenvolvimento da mastite, a fim de contribuir para melhor entendimento de processos biológicos, de componente celular e função molecular e vias biológicas envolvidas com a expressão desta característica. Quatro vacas foram abatidas e os úberes foram colhidos, perfundidos e inoculados com S. agalactiae. Para cada úbere, dois quartos foram inoculados (anterior esquerdo - AE; e posterior esquerdo - PE) e dois quartos foram utilizados como controle (anterior direito - AD; e posterior direito - PD). Biópsias foram feitas do tecido alveolar nos tempos 0 e 3 horas após a perfusão do tecido. O RNA das amostras foi extraído e sequenciado com a plataforma HiSeq2000 (Illumina). A partir das leituras obtidas os transcritos foram montados utilizando como referência a anotação do genoma bovino UMD 3.1 e então foram avaliadas quanto à sua expressão diferencial e funcionalidade. Métodos que assumem distribuição binomial negativa executados pelos métodos computacionais edgeR e baySeq e o método que assume distribuição beta binomial negativa executado pelo Cuffdiff, foram utilizados para análise de expressão gênica diferencial. Foram identificados 5.158 genes provenientes da união dos resultados obtidos com os três métodos computacionais. Esses genes foram investigados pela plataforma DAVID revelando o enriquecimento de termos de ontologia gênica, dentre os quais destacaram-se os processos de resposta a estímulos, sistema imune e processos apoptóticos. O perfil de genes diferencialmente expressos em amostras afetadas foi associado com a diferenciação, proliferação, migração e apoptose celular, desempenhando importante papel no local da inflamação por células do sistema imune, além de ativar vias de sinalização relacionadas ao sistema imune que são importantes nas primeiras três horas de infecção. Foram identificados apenas 122 genes com expressão diferencial em comum pelos três métodos utilizados. A restrição causada pelo uso da intersecção dos resultados obtidos com os três métodos não se mostrou adequada para realizar o enriquecimento funcional pois muitos genes de interesse foram desconsiderados e perdeu-se suporte estatístico nas análises de enriquecimento funcional. Assim, sugerimos que para esse tipo de estudo é mais adequado a utilização dos resultados de um dos métodos ou a união dos resultados dos três métodos. Este estudo permitiu maior entendimento das bases genéticas do desenvolvimento da mastite em úbere extracorpóreo de bovinos leiteiros mestiços holandês-Zebu, infectado experimentalmente por S. agalactiae, durante as primeiras horas de infecção.

Mastitis is responsible for huge economic losses in dairy cattle production which requires a better understanding of its genetic bases in order that genetic breeding methods could be developed. Therefore, some gene expression studies have been developed. The aims of this study were: 1. To detect, through three different computational methods, the differential gene expression data obtained by RNA sequencing from mammary glands of Holstein-Zebu crossbred dairy cattle, kept in an extracorporeal system and experimentally infected with Streptococcus agaclatiae; 2. To study the total gene expression profile and those genes common to the three methods, related with mastitis, in order to contribute to a better understanding of biological processes, cellular components, molecular functions and metabolic pathways related with the expression of this trait. Four cows were slaughtered and its udders were perfused and inoculated with S. agalactiae. For each udder, 2/4 were inoculated (front left - AE, and rear left - PE) and 2/4 were used as control (front right - AD, and rear right - PD). Biopsies were obtained from the alveolar tissue at 0 and 3 hours after the tissue perfusion. The RNA sample was extracted and sequenced with HiSeq 2000 platform (Illumina). From the reads obtained transcripts were assembled using as the reference the bovine genome annotation UMD 3.1, and then were evaluated for differential expression and functionality. Methods that assumes negative binomial distribution performed by edgeR and baySeq and the method that assumes beta negative binomial distribution performed by Cuffdiff were used for differential gene expression analysis. From the union of the results obtained with all computational methods, a total of 5,158 genes were identified. These genes were investigated by DAVID platform showing the enrichment of gene ontology terms, which were highlighted the response processes stimuli, immune response and apoptotic processes. The profile of differentially expressed genes identified in the affected samples was associated with differentiation, proliferation, migration and apoptosis, playing a major role in the inflammation site by immune system cells and in the activation of signaling pathways related to the immune system, which are important in the early three hours of infection. Only 122 differentially expressed genes were identified in common by the three methods. The restriction caused by the use of the intersection of the results obtained with the three methods was not interesting to perform functional enrichment analysis because there is loss of many interesting genes and the functional enrichment analysis has no statistical support. Thus, for this kind of study, we suggest that could be most appropriate the use of the results obtained with one method or the union of the results obtained with more than one method. This study allowed a better understanding of the genetic basis of mastitis developing in extracorporeal udder of Holstein-Zebu crossbred dairy cattle, during the early hours of experimentally infection by S. agalactiae.

PREDICTION OF GENOMIC-ENABLED BREEDING VALUES AND GENOME-WIDE ASSOCIATION STUDY FOR FEEDLOT AVERAGE DAILY WEIGHT GAIN IN NELORE CATTLE

Selection for fast growth rates using number of days to achieve specific weights or average weight gain would result in shorter production periods. Maintaining the rate of productivity increasing will demand, among other factors, genetically improved animals in both pasture and feedlot systems. Besides, genomic information could be used to predict genomic-enabled breeding values (GEBVs) earlier in animals' life, which would reduce generation intervals and increase productivity gains. Numerous studies have been conducted in order to identify appropriate methodologies to specific breeds and traits, which will result in more accurate GEBVs. The aim of this study was to compare the prediction accuracy of GEBVs and the ability to identify genomic regions and genes related to average weight daily gain in Nelore cattle, by applying different regression models and genotypes densities datasets. Genomic and phenotypic information of 804 steers born in three season, offspring of 34 bulls, were used to predict GEBVs through three models (Bayesian GBLUP, BayesA and BayesC ), four genotypic densities (Illumina BovineHD BeadChip, TagSNPs, GeneSeek Genomic Profiler High (HDi) and Low (LDi) density indicus) and two adjusted phenotypes. Family structure was accounted by using principal component analysis. Animals were assigned either to training (seasons 1 and 2) or testing (season 3) subsets to perform the cross-validation analysis. Estimates of Pearson correlation, regression coefficients and mean squared errors were used to access accuracy, inflation and bias of the estimated GEBVs, respectively. Genome-wide association study (GWAS) was also performed on above datasets, however, results were compared based on model complexity (pD) and deviance information criterion (DIC). SNP effects greater that upper limit of the 99.9% highest posterior interval were considered significant and genes located 25kb up or downstream were assigned to them. These genes were then, submitted to functional annotation clustering using DAVID database. Overall, accounting for family structure did not affect accuracy, but it seems having an effect on bias and inflation of predictions. Regarding to prediction of GEBVs, BayesC models produced the less biased and inflated results for the considered adjusted phenotypes. Furthermore, results from Bos taurus indicus-based chips were as informative as those from Illumina BovineHD, which could be an alternative in implementing genomic selection at lower costs. In relation to GWAS, BayesC model fitted better to the data, except for TagSNPs. A total of 11, 32 and 236 genes were found for LDi, HDi and 770k datasets, respectively, and the functional annotation assigned 3 genes to 1 cluster (LDi), 4 genes to 1 cluster and 35 genes to 3 (770k) enriched clusters. Five genes in common were found through all datasets: SYT1 (BTA 5), EYA2 (BTA 13), ZFHX4, MATN2 and CPQ (BTA 14). In general, the phenotype is a complex trait controlled by many genes and the model BayesC , which accounts for this performs better. Further investigations should account for the influence of the relationship between training and testing dataset.

A seleção para taxa de crescimento utilizando o número de dias para atingir determinado peso ou ganho de peso médio resultaria em menores ciclos de produção. Manter o aumento da produtividade exige, entre outros fatores, a utilização de animais melhorados, tanto nos sistemas de pastagem quanto de confinamento. Além disso, as informações genômicas podem ser usadas para predizer os valores genéticos genômicos (GEBVs) mais cedo na vida dos animais, o que reduziria os intervalos de geração e aumentaria os ganhos de produtividade. Inúmeros trabalhos tem sido conduzidos para identificar metodologias apropriadas à determinadas raças e características, o que irá resultar em GEBVs mais acurados. Os objetivos deste estudo foram comparar a acurácia de predição dos GEBVs e a habilidade de identificar regiões genômicas e genes relacionados ao ganho de peso médio diário em bovinos da raça Nelore, pela aplicação de diferentes modelos de regressão e densidades genotípicas. Informações genômica e fenotípica de 804 novilhos nascidos em três safras, filhos de 34 touros, foram utilizadas para predizer GEBVs por meio de três modelos (Bayesian GBLUP, BayesA e BayesC ), quatro densidades genotípicas (Illumina BovineHD BeadChip, TagSNPs, GeneSeek indicus de alta (HDi) e baixa (LDi) densidades) e dois fenótipos ajustados. A estrutura de família foi considerada por meio da análise de componentes principais. Os animais foram distribuídos em subconjunto de treinamento (safras 1 e 2) ou validação (safra 3) para realização da análise de validação cruzada. Estimativas de correlação de Pearson, coeficientes de regressão e erro quadrado médio foram usados para avaliar acurácia, inflação e viés dos GEBVs estimados, respectivamente. O estudo de associação ampla do genoma (GWAS) também foi realizado nos mesmos conjuntos de dados, entretanto, os resultados foram comparados com base na complexidade do modelo (pD) e no critério de informação do desvio (DIC). Os SNPs com efeitos maiores que o limite superior do intervalo de maior densidade a posteriori, com 99,9% de probabilidade, foram considerados significativos e, genes distantes 25kb de ambos os lados do SNP, foram relacionados. Esses genes foram submetidos ao agrupamento de anotação funcional no banco de dados DAVID. Em geral, a inclusão de informação de estrutura de família no modelo não afetou a acurácia, mas parece ter tido efeitos no viés e inflação das predições. Em relação a predição dos GEBVs, o modelo BayesC produziu os resultados menos viesados e inflacionados para os fenótipos ajustados. Além disso, os resultados obtidos com painéis baseados em Bos taurus indicus foram tão informativos quanto aqueles provenientes do painel Illumina BovineHD, o que pode ser uma alternativa para implementar a seleção genômica com menores custos de genotipagem. Em relação a GWAS, o modelo BayesC foi o que se ajustou melhor aos dados, com exceção do conjunto de TagSNPs. O total de 11, 32 e 236 genes foram identificados usando os conjuntos de dados LDi, HDi e 770k, respectivamente, e a anotação funcional resultou em 1 grupo com 3 genes (LDi), 1 grupo com 4 genes (HDi) e 35 genes distribuídos em 3 grupos (770k). Foram observados cinco genes em comum entre os três conjuntos de dados: SYT1 (BTA 5), EYA2 (BTA 13), ZFHX4, MATN2 e CPQ (BTA 14). De maneira geral, o fenótipo é uma característica complexa controlada por muitos genes e o modelo BayesC que leva isso em consideração, teve melhores resultados. Estudos futuros poderiam considerar a influência do grau de relacionamento entre os animais das populações de treinamento e teste.