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Generation of an Integrated Karyotype of the Honey Bee

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Generation of an Integrated Karyotype of the Honey Bee GENERATION OF AN INTEGRATED KARYOTYPE OF THE HONEY BEE (Apis mellifera L.) BY BANDING PATTERN AND FLUORESCENT IN SITU HYBRIDIZATION A Dissertation by GILDARDO AQUINO PEREZ Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2007 Major Subject: Entomology GENERATION OF AN INTEGRATED KARYOTYPE OF THE HONEY BEE (Apis mellifera L.) BY BANDING PATTERN AND FLUORESCENT IN SITU HYBRIDIZATION A Dissertation by GILDARDO AQUINO PEREZ Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committeee, J. Spencer Johnston Committee Members, Craig Coates Robert N. Coulson David M. Stelly Head of Department, Kevin Heinz December 2007 Major Subject: Entomology iii ABSTRACT Generation of an Integrated Karyotype of the Honey Bee (Apis mellifera L.) by Banding Pattern and Fluorescent In Situ Hybridization. (December 2007) Gildardo Aquino Perez, B.S. Universidad Autónoma Chapingo; M.S., Colegio de Postgraduados, Texcoco, Estado de México, México Chair of Advisory Committee: Dr. J. Spencer Johnston To enhance the scientific utility and practical application of the honey bee genome and assign the linkage groups to specific chromosomes, I identified chromosomes and characterized the karyotype of the sequenced strain DH4 of the honey bee. The primary analysis of the karyotype and ideogram construction was based on banding and Fluorescence In Situ Hybridization (FISH) for rDNA detection. FISH confirmed two locations for the NOR on telomeric regions of chromosomes 6 and 12 plus an additional less frequent signal on chromosome 1, all three of which were confirmed with silver staining (AgNO3). 4’6-diamidino-2phenylindole (DAPI), and C- Banding methods were used to construct the primary ideograms that served as a basis to further identify the chromosomes and locate important structures. The primary map was compared with Giemsa banding, AgNO3-banding, Trypsin banding, and R-banding. The karyotype of the honey bee was established as two metacentric chromosomes (1 and 10), two submetacentric with ribosomal organizer (6 and 12), four submetacentric heterochromatic chromosomes (16, 15, 4 and 13), four euchromatic subtelocentric chromosomes (2, 8, 11 and 14) and four acrocentric chromosomes (3, 5, 7 and 9). In situ nick-translation banding methods were used to verify the heterochromatin distribution. The cytogenetic maps of the honey bee karyotype represented in the ideograms were subsequently used to place 35 mapped BACs (Solignac et. al. 2004) of Solignac’s BAC library. As the BACs hybridized to multiple sites, the mapping was based on strength and frequency of the signals. Location and position of the BACs was compared with iv those published in the different version of Map Viewer of the NCBI and BeeBase web sites. 10 BACs were confirmed with the last version of Map Viewer V4, 12 BACs were mapped based on high frequency and agreement with the earlier version of Map Viewer. 14 BACs were mapped as confirmed based on moderate frequency of the signal and agreement with the last version of MVV, most of these BACs hits as a secondary signal. v DEDICATION To my Children: Verónica Donají, Elisa Aurora, and Marco Antonio To my Wife: Bertha López Santiago To my Father, Luis Rey Aquino Figueroa, To my Mother, Elisa Aurora Pérez Bláz (†) To God, who is Hope for everyone who suffers from injustice, abandon and contempt; the Source of forgiveness and hope of new life for the hopeless and repentant; the Glory of those who even without knowing Him but because of their actions are righteous people. vi ACKNOWLEDGEMENTS I am very grateful to Drs. Kim Jeong-Soon and Nurul Islam-Faridi from the Dept. of Crop and Soil Sciences, Texas A&M University, College Station, TX, and Dr. M. Mandrioli from Dipartimento di Biologia Animale, Università di Modena, Italy, for their kind recommendations spread preparations, to Dr. P. Klein from the Institute for Plant Genomics and Biotechnology of Texas A&M University for providing the BDG- 512 rDNA clone, to Dr. Tanya Pankiew for providing the biological material for DNA extraction, to Danny Weaver Apiaries of Navasota, TX, for providing the DH4 strain drones, and to Dr Hong-bin Zhang of the Department of Plant and Soil Science of Texas A&M University for his valuable help in the DNA extraction and purification. My special thanks to Dr. David Stelly for his friendship and support in the microscope facilities, and comments and suggestions during the experimental and writing work, and Dr. Robert Coulson, Dr. Craig Coates, and Dr. Patricia Pietrantonio, current and former members of my committee, for their support, suggestions and commentaries on my work. For their friendship and important logistic support, I am grateful to Dr Benjamin Figueroa, Dr. Felix Gonzalez, Dr Nina M. Barcenas, Fransisco Morales, Erasmo Rubio, Ivan Ramirez, Alma Solís, and Beatriz Rodriguez. Special thanks go to Miss Caroline Jones for her important support to my family, including friendship and valuable teaching. I would like to express my appreciation to my father and mother (†), sisters and brothers, who with their hard work supported my education. Above all, my gratitude goes to my family, who are my life, the reason to face the challenges of every day. To all I missed, my apologies and thanks. I am deeply grateful and indebted to my advisor and Chair of my Committee, Dr J. Spencer Johnston and his wife Diane, for their remarkable approachability and patience. To Dr. Johnston, for his support as I pursued my PhD in the Department of Entomology of Texas A&M University, and who provided not only the important economic support through an assistantship but also by his constructive comments and suggestions to gain experience in my novel field of molecular cytogenetics. vii TABLE OF CONTENTS Page ABSTRACT .................................................................................................................. iii DEDICATION .............................................................................................................. v ACKNOWLEDGEMENTS .......................................................................................... vi TABLE OF CONTENTS ............................................................................................. vii LIST OF FIGURES ....................................................................................................... viii LIST OF TABLES ........................................................................................................ x CHAPTER I INTRODUCTION ................................................................................... 1 II CHARACTERIZATION OF THE KARYOTYPE OF DRONES OF STRAIN DH4 OF Apis mellifera L ................................. 9 Introduction ............................................................................................. 9 Materials and Methods ........................................................................... 12 Results ..................................................................................................... 17 Discussion ............................................................................................... 51 III MAPPING 35 CLONES FROM SOLIGNAC’S BAC LIBRARY ON THE KARYOTYPE OF HONEY BEE ......................... 57 Introduction ............................................................................................. 57 Materials and Methods ........................................................................... 60 Results ..................................................................................................... 64 Discussion ............................................................................................... 96 IV IN SITU NICK-TRANSLATION BANDING IN DH4 HONEY BEE DRONE CHROMOSOMES ............................................ 103 Introduction ............................................................................................. 103 Materials and Methods ........................................................................... 105 Results ..................................................................................................... 107 Discussion ............................................................................................... 116 V CONCLUSIONS AND FUTURE WORK ............................................. 120 REFERENCES ............................................................................................................. 125 APPENDIX .................................................................................................................. 150 VITA ............................................................................................................................ 186 viii LIST OF FIGURES FIGURE Page 1 Standard deviation for three files and stages of prophase ....................... 20 2 Means for (a) Arm ratio, (b) chromosome, (c) short and (d) long arm length of individual chromosome at four stages of prophase, and stained with DAPI .................................................................................... 24 3 Chromosome classification at different stages of prophase ..................... 27 4 Means for (a) Relative length and (b) arm ratio of chromosomes of honey bee stained with six methods ........................................................ 28 5 Number of bands detected in honey bee chromosomes using DAPI
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