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“Deciphering the Genetic Architecture of Prolificacy Related Traits in an Experimental Iberian X Meishan F2 Intercross”

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UNIVERSITAT AUTÒNOMA DE BARCELONA Departament de Ciència Animal i dels Aliments Facultat de Veterinària “Deciphering the genetic architecture of prolificacy related traits in an experimental Iberian x Meishan F2 intercross” Ingrid Balcells Ortega PhD Thesis June, 2012 Supervisors: Armand Sánchez and Anna Tomás El Dr. Armand Sánchez Bonastre, catedràtic del Departament de Ciència Animal i dels Aliments de la Universitat Autònoma de Barcelona i la Dra. Anna Tomás Sangenís, investigadora en la Fundació d'Investigació Sanitària de les Illes Balears de Mallorca CERTIFIQUEN: Que l’Ingrid Balcells Ortega ha realitzat sota la seva direcció el treball de recerca “Deciphering the genetic architecture of prolificacy related traits in an experimental Iberian x Meishan F2 intercross” per a obtenir el grau de doctora per la Universitat Autònoma de Barcelona. Que aquest treball s’ha dut a terme al Departament de Ciència Animal i dels Aliments de la Facultat de Veterinària de la Universitat Autònoma de Barcelona. Bellaterra, 11 de Maig de 2012 Dr. Armand Sánchez Bonastre Dra. Anna Tomás Sangenís ACKNOWLEDGEMENTS Durant la realització d’aquesta tesi, han sigut moltes les persones que m’han acompanyat, tan a nivell professional com personal. Totes elles han aportat el seu granet de sorra per a que aquest projecte hagi tirat endavant i han fet que pugui recordar aquesta etapa amb un gran somriure a la cara. Als meus directors de tesi, el doctor Armand Sánchez i la doctora Anna Tomás. Per tota la confiança que heu dipositat en mi, per tots els coneixements que m’heu transmès, per donar-me copets a l’esquena en els moments de més desànim (sobretot en aquests últims mesos) i per mil coses més. Seria una llista molt llarga tot el que us voldria agrair. De tot cor, mil GRÀCIES per tot! I, a l’Anna, vull agrair-li també la seva gran paciència i les seves correccions tant en els articles com en la tesi. A la doctora Anna Castelló, sens dubte la persona qui més ha contribuït en la meva formació científica, sobretot a nivell de laboratori. Gràcies per estar sempre disposada a donar un cop de mà i pels teus bons consells en tot moment. Llàstima que siguis de Cervera! Als doctors Sebastian Ramós, Josep Mª Folch, Jordi Jordana, Miguel Pérez-Enciso, Marcel Amills i Jesús Piedrafita per haver contribuït amb la meva formació com a científica amb els vostres cursos de màster i xerrades a peu de passadís. A totes les nenes, i ara també nen, del SVGM i VETGENOMICS. A l’Olga, a la Laura i a l’Elisenda per les bones estones compartides i pels seus pràctics consells, a l’Anna Mercadé per oferir sempre la seva ajuda, al David per les llargues converses dins i fora el laboratori i a la Griselda, la Rebeca, la Lorena i la Carme per transmetre sempre alegria i felicitat. Als meus companys que han passat a ser grans amics, al Xavi (els biotecnòlegs primer!), a l’Oriol, a la Natàlia i a la Vero. Són molts els bons moments que hem passat junts (al laboratori, de congressos, viatges, sopars, birres,...) i de tots m’enduc un gran record i que espero que continuïn. I, a l’Oriol, per haver compartit les alegries i les penes del miRNAs. Amb tu, el treball amb RNA ha estat més fàcil! ACKNOWLEDGEMENTS Als companys que ja no hi són però que han deixat la seva petjada en la meva carrera científica: a l’Anna O., a la Maribel, a l’Oscar, al Jordi E., a l’Abid, a la María S., a la Neus, al John, a la Irene i al Yang Bin. A tota la resta de companys (espero no deixar-me a ningú, si ho faig, és per culpa les presses!): Alí (per la teva amistat i per les converses interessants que hem mantingut), Arianna (per les divertides converses en els dinars), Anna E. (pels bons moments i per oferir-me sempre la teva ajuda), al Quim C. (per resoldre tots els meus dubtes en estadística, que no han sigut pocs!), a la Marimar (per aguantar-nos), a la Maria B. (per transmetre ànims i pel teu entusiasme), a la Betlem (per aportar fil musical i alegria en tots els moments), a la Marta (per les converses estadístiques), a la Cecília (pels teus somriures) i, a les noves generacions: a la Sarai, a l’Anna P., al Jordi C., al Yuliaxis, a l’Erika, a la Carola i al William, pel vostre suport i amistat, molta sort en les vostres tesis! A la Susanna Cirera, per haver-me acollit tan bé durant la meva estada a Copenhaguen i haver fet que aquesta hagi estat molt enriquidora per mi, tant a nivell professional com personal. I would like to acknowledge Agnieszka Podoloska to be friendly with me and teach me how Denmark social life is. Moreover, thanks to everybody from the Department of Basic Animal and Veterinary Science (University of Copenhagen) to help me during my stay there. A tots els investigadors del IRTA-UdL, i de l’INIA per les llargues sessions a l’escorxador i per la vostra col·laboració en el projecte de la meva tesi. A tota la meva colla targarina, pels molts bons moments viscuts junts, per la vostra incondicional amistat i per fer-me saber que puc comptar amb vosaltres. Sou genials!! També a tots els amics biotecnòlegs: a la Cinta, a la Jone, a l’Alba, al Roger, al Jofre, al Joan, al Pep, a la Bàrbara, a la Paula, a l’Alberto, a la Sara, al Santi, al Xavi, ... per compartir tants bons moments durant i després la carrera. A l’Eloi, per estar al meu costat en tot moment, per donar-me grans dosis d’ànims en tots els moments de crisi i per ensenya’m a creure més en mi!! Gràcies per TOT!!! ACKNOWLEDGEMENTS A tota la meva família i, a tu iaia, per transmetre’m el teu amor i per rebre’m sempre amb una gran alegria i una gran abraçada. Als meus pares, al Jaume i a l’Anna, per estar SEMPRE al meu costat i per haver fet de mi la persona que sóc avui. GRÀCIES!! També al Jaume, el meu germà, per la gran estima i amistat que ens uneix i, perquè tot i que ens fem grans, encara em deixa fer de germana gran. SUMMARY DECIPHERING THE GENETIC ARCHITECTURE OF PROLIFICACY RELATED TRAITS IN AN EXPERIMENTAL IBERIAN X MEISHAN F2 INTERCROSS Reproductive traits are of great interest in the swine industry to improve the pig efficiency production. In a previous study, several QTL affecting reproductive traits were identified in an Iberian (Ib) x Meishan (Me) F2 population (Noguera et al., 2009, Fernandez-Rodriguez et al., 2010, Rodriguez et al., 2005). In particular, two QTL for the number of piglets born alive (NBA) and the total number of piglets born (TNB) were located on porcine chromosomes 13 (SSC13) and SSC17 (Noguera et al., 2009). To identify genes responsible for the prolificacy QTL on SSC13, four candidate genes (ITIH1, ITIH3, ITIH4 and MUC4) were analysed. Analyses for ITIH gene cluster showed that these genes had an effect on NBA independent of litter size QTL. Results for MUC4 gene, located within the confidence interval of the QTL on SSC13, determined that a SNP within the MUC4 gene was associated with NBA and TNB although the effect was stronger for NBA. In addition, uterine MUC4 expression was two-fold higher in high prolificacy sows. To better understand the genetic basis of prolificacy related traits, transcriptome analyses, which included the study of uterine gene expression as well as the miRNA expression profile in the uterus and in the ovary, was performed. For this, IbxMe F2 sows displaying extreme phenotypes regarding the prolificacy levels defined as the number of embryos (NE) attached to the uterus at day 30-32 of the gestation were used. In uterus of high prolificacy sows, 101 genes involved in the inflammatory response to stimulus and muscle tissue development were upregulated whereas 196 genes that participated in muscle tissue development, cell junction organization and adhesion, biological regulation, muscle and circulatory system processes, and transport were downregulated. The microRNAome identified the miR-125b-5p, miR- 200c-3p, miR-23b-3p, miR-23a-3p and miR-99a-5p as the most abundant miRNAs in uterus of pregnant sows while expression of miR-139-5p, miR-150-5p, miR-27a-3p and miR-20-5p was associated with prolificacy levels. Among predicted gene target for uterine prolificacy-related miRNAs, 32 were located within the confidence interval of the QTL for NBA and TNB and therefore, they are proposed to be good candidate genes to be further investigated. Importantly, among these candidate genes, it is found MUC4 gene which is of great interest to be the responsible for the prolificacy QTL on SSC13 because it fulfilled several criteria: it is located within the QTL confidence interval with an effect on NBA and TNB, its expression level varies regarding the prolificacy level of sows, and it is targeted by miR-150-5p, a miRNA that was also found diferentially expressed regarding the prolificacy levels. On the other hand, ovarian miR-146a-5p and miR-142-3p, involved in immune system processes and cellular homeostasis, were differentially expressed regarding prolificacy levels. Four predicted target genes (LRRK1, CCL8, CPEB2 and BAT1), located within confidence intervals for prolificacy QTL, are good candidate genes to be studied for QTL on litter size. Alternatively, we have designed a new RT-qPCR methodology, by using DNA primers to measure miRNA expression, which is highly specific, sensitive and accurate.
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  • Evaluation of a New Method for Large-Scale and Gene-Targeted Next Generation DNA Sequencing in Nonmodel Species

    Evaluation of a New Method for Large-Scale and Gene-Targeted Next Generation DNA Sequencing in Nonmodel Species

    University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2013 Evaluation of a New Method for Large-Scale and Gene-targeted Next Generation DNA Sequencing in Nonmodel Species Ted Cosart The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Cosart, Ted, "Evaluation of a New Method for Large-Scale and Gene-targeted Next Generation DNA Sequencing in Nonmodel Species" (2013). Graduate Student Theses, Dissertations, & Professional Papers. 4133. https://scholarworks.umt.edu/etd/4133 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. EVALUTATION OF A NEW METHOD FOR LARGE-SCALE AND GENE- TARGETED NEXT GENERATION DNA SEQUENCING IN NONMODEL SPECIES By Ted Cosart BA, University of Montana, Missoula, Montana, 1983 MS, University of Montana, Missoula, Montana, 2006 Dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Individualized, Interdisciplinary Graduate Program The University of Montana Missoula, Montana August, 2013 Approved by: Sandy Ross, Associate Dean of The Graduate School Graduate School Dr. Jesse Johnson, Co-Chair Computer Science Dr. Gordon Luikart, Co-Chair Flathead Biological Station Dr. Jeffrey Good Division of Biological Sciences Dr. William Holben Division of Biological Sciences Dr. Stephen Porcella Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases Dr.