Application of molecular markers on genetic improvement and reproduction in the post-genomic Era: Emerging biotechnologies and perspectives in livestock

Background: New challenges are rising in the animal protein market, and one of the main world challenges is to produce more in shorter time, with better quality and in a sustainable way. Brazil is the largest beef exporter in volume hence the factors affecting the beef meat chain are of major concern in countrýs economy. An emerging class of biotechnological approaches, the molecular markers, is bringing new perspectives to face these challenges, particularly after the publication of the first complete livestock genome (bovine), which has triggered a massive initiative to put in practice the benefits of the so called the Post-Genomic Era. Review: This article aimed at showing the directions and insights in the application of molecular markers on livestock genetic improvement and reproduction as well at organizing the progress so far, pointing some perspectives of these emerging technologies in Brazilian ruminant production context. An overview on the nature of the main molecular markers explored in ruminant production is provided, which describes the molecular bases and detection approaches available for microsatellites (STR) and single nucleotide polymorphisms (SNP). A topic is dedicated to review the history of association studies between markers and important trait variation in livestock, showing the timeline starting on quantitative trait loci (QTL) identification using STR markers and ending in high resolution SNP panels to proceed whole genome scans for phenotype/genotype association. Also the article organizes this information to reveal how QTL prospection using STR could open ground to the feasibility of marker-assisted selection and why this approach is quickly being replaced by studies involving the application of genome-wide association using SNP research in a new concept called genomic selection. Conclusion: The world's scientific community is dedicating effort and resources to apply SNP information in livestock selection through the development of high density panels for genomic association studies, connecting molecular genetic data with phenotypes of economic interest. Once generated, this information can be used to take decisions in genetic improvement programs by selecting animals with the assistance of molecular markers.

Influence of optimized lubrication-cooling and minimum quantity lubrication on the cutting forces, on the geometric quality of the surfaces and on the micro-structural integrity of hardened steel parts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

How bioinformatics enables livestock applied sciences in the genomic era

This review paper presents the three main approaches currently used in livestock genomic sciences where the bioinfomatics plays a critical role. They are named as Genomic Selection (GS), Genome Wide Association Study (GWAS) and Signatures of Selection (SS). The subsides for the construction of this article were generated in a current project (started in 2011), so called Zebu Genome Consortium (ZGC), which joins researchers from different institutions and countries, aiming to scientifically explore genomic information of Bos taurus indicus cattle breeds and deliver useful information to breeders and academic community, specially from the tropical regions of the world. © 2012 Springer-Verlag.

Chromosome mapping of repetitive sequences in Anostomidae species: Implications for genomic and sex chromosome evolution

Background: Members of the Anostomidae family provide an interesting model system for the study of the influence of repetitive elements on genome composition, mainly because they possess numerous heterochromatic segments and a peculiar system of female heterogamety that is restricted to a few species of the Leporinus genus. The aim of this study was to isolate and identify important new repetitive DNA elements in Anostomidae through restriction enzyme digestion, followed by cloning, characterisation and chromosome mapping of this fragment. To identify repetitive elements in other Leporinus species and expand on studies of repetitive elements in Anostomidae, hybridisation experiments were also performed using previously described probes of LeSpeI repetitive elements. Results: The 628-base pair (bp) LeSpeII fragment was hybridised to metaphase cells of L. elongatus individuals as well as those of L. macrocephalus, L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii and S. isognathus. In L. elongatus, both male and female cells contained small clusters of LeSpeII repetitive elements dispersed on all of the chromosomes, with enrichment near most of the terminal portions of the chromosomes. In the female sex chromosomes of L. elongatus (Z2,Z2/W1W 2), however, this repeated element was absent. In the remaining species, a dispersed pattern of hybridisation was observed on all chromosomes irrespective of whether or not they were sex chromosomes. The repetitive element LeSpeI produced positive hybridisations signals only in L. elongatus, L. macrocephalus and L. obtusidens, i.e., species with differentiated sex chromosomes. In the remaining species, the LeSpeI element did not produce hybridisation signals. Conclusions: Results are discussed in terms of the effects of repetitive sequences on the differentiation of the Anostomidae genome, especially with respect to sex chromosome evolution. LeSpeII showed hybridisation patterns typical of Long Interspersed Elements (LINEs). The differential distribution of this element may be linked to sex chromosome differentiation in L. elongatus species. The relationship between sex chromosome specificity and the LeSpeI element is confirmed in the species L. elongatus, L. macrocephalus and L. obtusidens. © 2012 da Silva et al.; licensee BioMed Central Ltd.

Evaluation of Sperm Kinetics and Plasma Membrane Integrity of Frozen Equine Semen in Different Storage Volumes and Freezing Conditions

In the present study, different freezing systems (Styrofoam box and Mini Digitcool ZH 400) and storage volumes (0.5- and 0.25-mL straws) were compared with regard to sperm kinetics and plasma membrane integrity of frozen and thawed semen. For that, three ejaculates from four animals were frozen in Styrofoam box and Mini Digitcool ZH 400 machine. The 0.5-mL straws were thawed at 46°C for 20 seconds, and the 0.25-mL straws were thawed at 46°C for 12 seconds. Statistical analysis was performed using program R of descriptive analysis box plot, followed by analysis of variance using PROC MIXED of SAS 9.1 package. Variances of 5% were considered as different. There was no interaction between the straw sizes and volumes; however, statistical differences were observed between the semen storage volumes. The 0.5-mL straws had higher total motility (%), progressive motility (%), average path velocity (μm/s), straight-line velocity (μm/s), curvilinear velocity (μm/s), and rapid sperm percentage (%) than the 0.25-mL straws. However, plasma membrane integrity analysis did not differ between the two straws. Thus, it is possible to conclude that equine sperm cryopreserved in 0.5-mL straws has better sperm kinetics than when stored in 0.25-mL straws. Additionally, it is possible to conclude that automated systems that enable faster freezing rates result in a seminal quality that is similar to the one obtained by the conventional system using Styrofoam boxes. © 2013 Elsevier Inc.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.

Morphological and Genomic Differences Between Cutting and Racing Lines of Quarter Horses

To investigate morphological and genomic differences between cutting and racing lines of Quarter Horses, 120 racing and 68 cutting animals of both sexes, registered at the Brazilian Association of Quarter Horse Breeders, were used. Blood samples were collected, and the following physical traits were measured: weight; height at withers; body length; length of the shank, pastern, rump, head, and neck; and chest, shank, and hoof circumference. For analysis of genomic differences, 54,602 single-nucleotide polymorphisms (SNPs) were genotyped using the Equine SNP50 BeadChip, and the quality of individual and SNP genotype data were evaluated. The fixation index, FST, was used to identify genome regions that were altered in the lines by selection. The results showed significant differences between the lines in all physical traits. Quality control led to the exclusion of four cutting animals with a call rate of <0.95. After filtering, 12,544, 13,815, and 13,370 SNPs were excluded for the whole population (n = 184), the 120 racing animals, and the 64 cutting animals, respectively. The number of informative polymorphisms detected in each line and in the whole population indicated that the Equine SNP50 BeadChip can be used in genetic studies of Quarter Horses. The fixation index, FST, identified 2,558 genome regions that may have been modified by divergent selection. © 2013 Elsevier Inc.

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.

Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research. © 2013 Spandidos Publications Ltd. All rights reserved.

Screening of genomic libraries

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs. © Springer Science+Business Media, LLC 2013.

Effects of corn replacement by sorghum in broiler diets on performance and intestinal mucosa integrity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genomic Signatures Predict Poor Outcome in Undifferentiated Pleomorphic Sarcomas and Leiomyosarcomas

Undifferentiated high-grade pleomorphic sarcomas (UPSs) display aggressive clinical behavior and frequently develop local recurrence and distant metastasis. Because these sarcomas often share similar morphological patterns with other tumors, particularly leiomyosarcomas (LMSs), classification by exclusion is frequently used. In this study, array-based comparative genomic hybridization (array CGH) was used to analyze 20 UPS and 17 LMS samples from untreated patients. The LMS samples presented a lower frequency of genomic alterations compared with the UPS samples. The most frequently altered UPS regions involved gains at 20q13.33 and 7q22.1 and losses at 3p26.3. Gains at 8q24.3 and 19q13.12 and losses at 9p21.3 were frequently detected in the LMS samples. Of these regions, gains at 1q21.3, 11q12.2-q12.3, 16p11.2, and 19q13.12 were significantly associated with reduced overall survival times in LMS patients. A multivariate analysis revealed that gains at 1q21.3 were an independent prognostic marker of shorter survival times in LMS patients (HR = 13.76; P = 0.019). Although the copy number profiles of the UPS and LMS samples could not be distinguished using unsupervised hierarchical clustering analysis, one of the three clusters presented cases associated with poor prognostic outcome (P = 0.022). A relative copy number analysis for the ARNT, SLC27A3, and PBXIP1 genes was performed using quantitative real-time PCR in 11 LMS and 16 UPS samples. Gains at 1q21-q22 were observed in both tumor types, particularly in the UPS samples. These findings provide strong evidence for the existence of a genomic signature to predict poor outcome in a subset of UPS and LMS patients. © 2013 Silveira et al.

Effects of pre-slaughter fasting on broiler welfare, meat quality, and intestinal integrity

The Brazilian Ministry of Agriculture (MAPA) regulations establish 12 hours as the maximum pre-slaughter fasting period for broilers; however, many processing plants have considered this time is not sufficient, and consequently return the birds to the farms, with consequent economic losses and welfare problems. Therefore, it is necessary to investigate the possible effects of longer pre-slaughter fasting times. The objective of the present study was to evaluate the effect of pre-slaughter fasting times longer than those established by MAPA on broiler welfare, breast meat quality, and intestinal integrity. Forty 42-d-old broilers were submitted to different pre-slaughter fasting times: group I: 6 hours, group II 9h, group III 12h, and group IV 15h. Bird welfare was assessed before slaughter. After sacrifice, intestinal samples were collected to assess their morphology and morphometrics, and the Pectoralis major muscle was analyzed for pH and color. There was no influence (p>0.05) of treatments on breast muscle pH or color.There were no significant changes in intestinal morphometrics (p<0.05). Bird behavior was affected (p<0.05), suggesting that welfare was impaired as fasting time increased, but no differences in the analyzed parameters were detected between broilers fasted for 12 or 15 hours. It was concluded that the behavioral differences between birds fasted for 12 and 15 hours are not sufficient to assert that those fasted for 15 hours were in worse welfare conditions.

Caracterização de uma região genômica relacionada a uma anomalia de viveiro em Eucalyptus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência do método de separação dos espermatozóides viáveis (swim-up) na eficiência de seleção do sexo de bovinos por gradiente descontínuo de densidade e o impacto no melhoramento genético animal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)