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Molecular Evolution of MADS-Box Genes in Cotton (Gossypium L.)

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Molecular Evolution of MADS-Box Genes in Cotton (Gossypium L.) University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2007 Molecular Evolution of MADS-box Genes in Cotton (Gossypium L.) Wusheng Liu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Botany Commons, and the Plant Breeding and Genetics Commons Recommended Citation Liu, Wusheng, "Molecular Evolution of MADS-box Genes in Cotton (Gossypium L.). " PhD diss., University of Tennessee, 2007. https://trace.tennessee.edu/utk_graddiss/231 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Wusheng Liu entitled "Molecular Evolution of MADS-box Genes in Cotton (Gossypium L.)." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Botany. Randall Small, Major Professor We have read this dissertation and recommend its acceptance: Karen Hughes, Barry Bruce, Beth Mullin Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting here with a dissertation written by Wusheng Liu entitled “Molecular Evolution of MADS-box genes in Cotton (Gossypium L.)”. I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Botany. Randall Small Major Professor We have read this dissertation and recommend its acceptance: Karen Hughes Barry Bruce Beth Mullin Acceptance for the Council: Linda Painter Interim Dean of Graduate Studies (Original signatures are on file with official student records.) MOLECULAR EVOLUTION OF MADS-BOX GENES IN COTTON (Gossypium L.) A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Wusheng Liu May 2007 DEDICATION This dissertation is dedicated to my dear mother, Shuqin Xu, who passed away when I was seven years old, and my dear father, Yonghui Liu, who passed away when I was a freshman in college, and my brother, Zaisheng Liu, for always caring about me, educating me, and encouraging me to achieve my goals. ii ACKNOWLEDGEMENTS I wish to thank all those who helped me in completing my doctoral degree in Botany. Special thanks go to my committee chair, Dr. Small, and to the dissertation committee members Dr. Bruce, Dr. Hughes and Dr. Mullin, for their inspiring guidance and efforts through this research. I would like to thank Dr. Nebenführ and Dr. von Arnim for valuable discussions and ideas for this project. I would also like to thank Dr. McFarland for taking care of my plants growing in the greenhouse, and Dr J. Wendel (Iowa State University) for providing flower buds of Gossypium raimondii. Lastly, I would like to thank all the people in former Botany Department, and Ecology and Evolutionary Biology Department, for teaching, being friends and providing assistantships. iii ABSTRACT The purpose of this study was to examine the molecular evolution of floral developmental MADS-box genes in diploid and allotetraploid cotton (Gossypium, Malvaceae). We isolated and characterized both cDNA and genomic DNA sequences of four MADS-box genes, B-sister, APETALA3 (AP3), PISTILLATA (PI, two copies) and AGAMOUS (AG), in seven Gossypium species (2 A-genome species, 2 D-genome species, 1 C-genome species and 2 AD-genome species) as well as an outgroup species, Gossypioides kirkii. We then studied the expression patterns of each gene by reverse- transcriptase PCR (RT-PCR) in each flower part for different flower stages of G. arboreum (A-genome), G. raimondii (D-genome) and G. hirsutum (AD-genome). We found that these MADS-box genes are evolving independently at the DNA sequence level but exhibit highly variable expression patterns in allotetraploid G. hirsutum. These MADS-box genes contain highly conserved exon lengths and exon/intron structures, high nucleotide identity with homologs in Arabidopsis thaliana and Antirrhinum majus, and conserved gene phylogeny within the genus Gossypium. All the genes exhibit evidence of codon bias. The AP3 gene in Gossypium appears to be subject to positive selection, while the PI-small copy of the PI gene appears to be subject to strong functional constraints together with a very low mutation rate. Within G. hirsutum, the B-sister gene exhibits gene silencing of the A-subgenomic homoeolog and the PI-big copy of the PI gene has experienced pseudogenization by insertion of a copia-like retroelement. The AP3, PI-small and AG genes show biased expression patterns in G. hirsutum. These MADS-box genes also demonstrated developmentally regulated expression patterns in G. arboreum, G. raimondii and G. hirsutum. iv TABLE OF CONTENTS CHAPTER 1...................................................................................................................... 2 INTRODUCTION............................................................................................................. 2 Part I MADS-box gene family........................................................................................ 2 1. The genetic hierarchy of flower pattern formation process.................................... 2 2. Floral organ identity genes...................................................................................... 2 (1) Phylogeny of all the MADS-box genes in plants and animals.......................... 4 (2) Type II plant MADS-box genes--- MIKCc- and MIKC*-type genes ................ 4 3. MIKCc-type MADS-box genes............................................................................... 6 (1) Models of the expression patterns of MIKCc-type genes for floral organ identity .................................................................................................................... 6 (2) Phylogeny of MIKCc-type genes ...................................................................... 7 (3) Evolution of MIKCc-type genes...................................................................... 13 (4) Summary and future prospects........................................................................ 15 Part II Polyploidy.......................................................................................................... 16 1. Initial stages of polyploidy formation................................................................... 17 (1) Cytonuclear stabilization following polyploidization..................................... 17 (2) Rapid genetic changes..................................................................................... 17 (3) Rapid epigenetic changes................................................................................ 18 2. Longer-term genic and genomic evolution........................................................... 19 (1) Longer-term genic evolution........................................................................... 19 (2) Longer-term genomic evolution...................................................................... 22 Part III The Genus of Gossypium L. ............................................................................. 24 1. Phylogeny of the genus Gossypium L................................................................... 24 (1) Genealogical framework and main divergence of genus Gossypium ............. 24 (2) Possible origin and dispersal pathways of the genus of Gossypium ............... 27 2. Molecular Evolution of genes and genomes in Gossypium.................................. 29 (1) Genomic Evolution in the genus Gossypium .................................................. 29 (2) Allopolyploidy-induced Genic Evolution in the tetraploid Gossypium.......... 30 CHAPTER II................................................................................................................... 33 MATERIALS AND METHODS ................................................................................... 33 Part I Plant Materials .................................................................................................... 33 Part II Methods ............................................................................................................. 34 1. Cloning and sequencing of MADS-box genes...................................................... 34 (1) RNA extraction ............................................................................................... 34 v (2) Isolation of cDNAs of MADS-box domains by RT-PCR, Cloning and Sequencing............................................................................................................ 34 (3) Isolation of the whole cDNAs and genomic sequences of each gene by 5´/ 3´ RACE and PCR amplification followed by cloning and sequencing ................... 35 2. Gene structure analyses......................................................................................... 36 3. Expression pattern studies by RT-PCR................................................................. 36 4. Phylogenetic analysis...........................................................................................
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