Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B. bubalis) chromosome 20

<p>Abstract</p> <p>Background</p> <p>Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method.</p> <p>Results</p> <p>As proof of principle for this dissociation curve method, we generated new maps of river buffalo (<it>Bubalus bubalis</it>) chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly.</p> <p>Conclusion</p> <p>Dissociation curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based dissociation curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.</p

Differential Gene Expression in High- and Low-Active Inbred Mice

Numerous candidate genes have been suggested in the recent literature with proposed roles in regulation of voluntary physical activity, with little evidence of these genes' functional roles. This study compared the haplotype structure and expression profile in skeletal muscle and brain of inherently high- (C57L/J) and low- (C3H/HeJ) active mice. Expression of nine candidate genes [Actn2, Actn3, Casq1, Drd2, Lepr, Mc4r, Mstn, Papss2, and Glut4 (a.k.a. Slc2a4)] was evaluated via RT-qPCR. SNPs were observed in regions of Actn2, Casq1, Drd2, Lepr, and Papss2; however, no SNPs were located in coding sequences or associated with any known regulatory sequences. In mice exposed to a running wheel, Casq1 (P = 0.0003) and Mstn (P = 0.002) transcript levels in the soleus were higher in the low-active mice. However, when these genes were evaluated in naïve animals, differential expression was not observed, demonstrating a training effect. Among naïve mice, no genes in either tissue exhibited differential expression between strains. Considering that no obvious SNP mechanisms were determined or differential expression was observed, our results indicate that genomic structural variation or gene expression data alone is not adequate to establish any of these genes' candidacy or causality in relation to regulation of physical activity

The role of “omics” technologies for livestock sustainability

Un exitoso programa de seguridad alimentaria de una población en crecimiento necesita una producción más eficiente y sostenible del ganado para asegurar la disponibilidad de alto valor nutritivo, y también la salud y el bienestar de los seres humanos y los animales, y protección del medio ambiente. Innovaciones y avances tecnológicos vendrán de la investigación básica y fundamental. En particular, la integración de la genómica con otros campos de investigación '' OMIC '' y métodos computacionales, posiblemente, facilitar la comprensión de los mecanismos biológicos que son responsables de los atributos físicos o fenotipos de animales. Ejemplos OMIC de diversos métodos y sus aplicaciones se describen para demostrar los avances recientes y cómo estos métodos se pueden aplicar a la producción animal. Los avances en la investigación en genómica y otros campos ómicas se pueden utilizar para aumentar la productividad de los animales, satisfacer la creciente demanda de alimentos de origen animal, aumentar la disponibilidad de nutrientes de alta calidad, garantizar la seguridad alimentaria, mitigar los efectos de la variabilidad climático, y el resultado en nuevas tecnologías que favorezcan un mejor seguridad de los alimentos en todo el mundo.A successful program of food security for a growing population requires the production of livestock in the most efficient and sustainable manner possible to ensure the availability of nutritious foods, the overall health and well-being of humans and animals, and the protection of the environment. Innovative and technological advancements that enhance all aspects of food production will arise from basic, fundamental research. Notably, the integration of genomics with other “’omics” research fields and computational methods will continue to lead to a better understanding of biological mechanisms that are responsible for physical attributes, or phenotypes. Examples of several ‘omics methods and their applications are described to demonstrate recent advances and how these methods can be applied to livestock. Research breakthroughs in genomics and other ‘omics fields can be used to enhance the productivity of food animals, meet the increasing demand for animal-sourced foods, enhance high-quality nutrient availability, ensure nutrient safety, mitigate the effects of climate variability, and result in new technologies that provide continuous improvement in food security worldwide.Um programa bem-sucedido de segurança alimentar para uma população em crescimento necessita de uma produção animal mais eficiente e sustentável para assegurar a disponibilidade de alimentos de alto valor nutricional, e também a saúde e o bem-estar dos humanos e dos animais, e a proteção do ambiente. Inovações e avanços tecnológicos surgirão da pesquisa básica e fundamental. Em especial, a integração da genômica com outros campos de pesquisa ''ômicos '' e com métodos computacionais possivelmente facilitará o entendimento dos mecanismos biológicos que são responsáveis por atributos físicos ou fenótipos dos animais. Exemplos de vários métodos ômicos e suas aplicações são descritos para demonstrar os avanços recentes e como esses métodos podem ser aplicados para produção animal. Os avanços na pesquisa em genômica e em outros campos ômicos podem ser usados para aumentar a produtividade de animais, atender à crescente demanda por alimentos de origem animal, aumentar a disponibilidade de nutrientes de alta qualidade, garantir a segurança nutricional, mitigar os efeitos da variabilidade climática, e resultar em novas tecnologias que favorecem a melhora a segurança alimentar em todo o mundo

Differential Gene Expression in High- and Low-Active Inbred Mice

Numerous candidate genes have been suggested in the recent literature with proposed roles in regulation of voluntary physical activity, with little evidence of these genes’ functional roles. This study compared the haplotype structure and expression profile in skeletal muscle and brain of inherently high- (C57L/J) and low- (C3H/HeJ) active mice. Expression of nine candidate genes [Actn2, Actn3, Casq1, Drd2, Lepr, Mc4r, Mstn, Papss2, and Glut4 (a.k.a. Slc2a4)] was evaluated via RT-qPCR. SNPs were observed in regions of Actn2, Casq1, Drd2, Lepr, and Papss2; however, no SNPs were located in coding sequences or associated with any known regulatory sequences. In mice exposed to a running wheel, Casq1 (P=0.0003) and Mstn (P=0.002) transcript levels in the soleus were higher in the low-active mice. However, when these genes were evaluated in naïve animals, differential expression was not observed, demonstrating a training effect. Among naïve mice, no genes in either tissue exhibited differential expression between strains. Considering that no obvious SNP mechanisms were determined or differential expression was observed, our results indicate that genomic structural variation or gene expression data alone is not adequate to establish any of these genes’ candidacy or causality in relation to regulation of physical activity

Evolutionary diversity and developmental regulation of X-chromosome inactivation

X-chromosome inactivation (XCI) results in the transcriptional silencing of one X-chromosome in females to attain gene dosage parity between XX female and XY male mammals. Mammals appear to have developed rather diverse strategies to initiate XCI in early development. In placental mammals XCI depends on the regulatory noncoding RNA X-inactive specific transcript (Xist), which is absent in marsupials and monotremes. Surprisingly, even placental mammals show differences in the initiation of XCI in terms of Xist regulation and the timing to acquire dosage compensation. Despite this, all placental mammals achieve chromosome-wide gene silencing at some point in development, and this is maintained by epigenetic marks such as chromatin modifications and DNA methylation. In this review, we will summarise recent findings concerning the events that occur downstream of Xist RNA coating of the inactive X-chromosome (Xi) to ensure its heterochromatinization and the maintenance of the inactive state in the mouse and highlight similarities and differences between mammals

Differential Expression of MicroRNAs in Dark-Cutting Meat from Beef Carcasses

&ldquo;Dark-cutting&rdquo; meat in beef carcasses can result from conditions such as long-term stress and depleted glycogen stores, but some aspects of the physiological mechanisms that cause dark-cutting phenotypes remain poorly understood. Certain responses to stress factors in fully developed tissues are known to be regulated by specific microRNAs. We investigated microRNA expression in Longissimus lumborum biopsies from carcasses derived from a contemporary group of 78 steers from which a high incidence of dark-cutting meat occurred. Our objective was to identify any potential microRNA signatures that reflect the impact of environmental factors and stresses on genetic signaling networks and result in dark-cutting beef (also known as dark, firm, and dry, or DFD) in some animals. MicroRNA expression was quantified by Illumina NextSeq small RNA sequencing. When RNA extracts from DFD muscle biopsy samples were compared with normal, non-DFD (NON) samples, 29 differentially expressed microRNAs were identified in which expression was at least 20% different in the DFD samples (DFD/NON fold ratio &le;0.8 or &ge;1.2). When correction for multiple testing was applied, a single microRNA bta-miR-2422 was identified at a false discovery probability (FDR) of 5.4%. If FDR was relaxed to 30%, additional microRNAs were differentially expressed (bta-miR-10174-5p, bta-miR-1260b, bta-miR-144, bta-miR-142-5p, bta-miR-2285at, bta-miR-2285e, bta-miR-3613a). These microRNAs may play a role in regulating aspects of stress responses that ultimately result in dark-cutting beef carcasses