Copyright by Jin Zhang 2015 The Dissertation Committee for Jin Zhang Certifies that this is the approved version of the following dissertation: Coevolution between Nuclear and Plastid Genomes in Geraniaceae Committee: Robert K. Jansen, Supervisor David L. Herrin C. Randy Linder Claus O. Wilke Stanley J. Roux Coevolution between Nuclear and Plastid Genomes in Geraniaceae by Jin Zhang, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August 2015 Acknowledgements I would like to express my greatest appreciation to my wonderful supervisor, Dr. Robert K. Jansen, for giving me the opportunity to work in his lab, allowing me the freedom for exploration and making mistakes, being the ultimate supporter whenever I needed it, and motivating me every day to become a better scientist. I also want to thank Dr. Tracey Ruhlman for all the inspiration in my research, comprehensive guidance on my experimental work and my scientific writing skills, and I want to thank our collaborator Dr. Jeffery Mower for his guidance in bioinformatics techniques and the accommodations for my short visit to University of Nebraska Lincoln. Additionally, I would like to express my appreciation to my committee (Dr. David Herrin, Dr. Randy Linder, Dr. Claus Wilke and Dr. Stanley Roux) for their support, time and valuable suggestions. I want to thank my long standing lab mates Dr. Chris Blazier, Maolun Weng, Erika Schwarz, Anna Yu and Dr. Seongjun Park for the inspiring discussions and being incredible resources of plant biology and evolutionary knowledge; Dr. Michael Gruenstaeudl for the help in my cloning experiments; Dhivya Arasappan for the help in scripting in my early PhD life; the Texas Advanced Computing Center for their valuable and patient assistance. I also want to thank all my friends during my PhD life, my best friend Dr. Shanshan Cao, who came to the USA with me in same year, for his incredible support and friendship; my five-year roommates, who are also my class mates, An Li, Zicheng Hu, Xianzhe Wang, Chao Liu and Dongyang Wang, for the inspiring discussion in research and the sharing of all the joyful moments in our lives. iv Lastly I would like to thank my family, my father Qingyu Zhang, my mother Feng Jin and my girlfriend Xiwei Yan, for their lifelong love, support and understanding during all these years. From them I learned how to be a man who is honest, supportive and hard working. v Coevolution between Nuclear and Plastid Genomes in Geraniaceae Jin Zhang, PhD The University of Texas at Austin, 2015 Supervisor: Robert K. Jansen Plastid genomes of angiosperms are highly conserved in both genome organization and nucleotide substitution rates. Geraniaceae have highly rearranged genomes and elevated nucleotide substitution rates, which provides an attractive system to study nuclear-plastid genome coevolution. My dissertation research has focused on two areas of nuclear-plastid genome coevolution in Geraniaceae. First, I have investigated the correlation of nucleotide substitution rates between nuclear and plastid genes that encode interacting subunits that form the multi-subunit complex of Plastid Encoded RNA Polymerase (PEP). Second, the hypothesis that the unusual changes of plastid genome organization and elevated nucleotide substitution rates of plastid encoded genes is the result of alterations in nuclear encoded DNA replication, recombination and repair (DNA RRR) genes is tested. The second chapter investigates the optimal methods for transcriptome sequencing/assembly. My findings supported the use of transcriptome assemblers optimized for Illumina sequencing platform (Trinity and SOAPdenovo-trans). The third chapter investigated coevolution of nucleotide substitution rates between plastid encoded RNAP (rpoA, rpoB, rpoC1, rpoC2) and nuclear encoded SIG (sig1-6) genes that are part of the multi-subunit complex PEP. Using the transcriptomes of 27 Geraniales species I extracted the PEP genes and performed a systematic correlation test. I detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous vi substitutions) between RNAP and SIG but no correlations were detected for the control genes, which provides a plausible explanation for the cause of plastome-genome incompatibility in Geraniaceae. The fourth chapter investigated the effect of DNA RRR system on the aberrant evolutionary phenomena in Geraniaceae plastid genomes. I extracted DNA RRR and nuclear control genes with different subcellular locations from 27 Geraniales transcriptomes and estimated genome complexity with various measures from plastid genomes of the same species. I detected significant correlations for dN but not dS for three DNA RRR genes, 10 nuclear encoded plastid targeted (NUCP) and three nuclear encoded mitochondrial targeted (NUMT) genes. The findings of a correlation between dN of DNA RRR genes and genome complexity support the hypothesis that changes of plastid genome complexity in Geraniaceae may be caused by dysfunction of DNA RRR systems. vii Table of Contents List of Tables ......................................................................................................... xi! List of Figures ...................................................................................................... xiii! Chapter 1: Introduction ............................................................................................1! Chapter 2: Comparative analyses of two Geraniaceae transcriptomes using next- generation sequencing .....................................................................................4! Background .....................................................................................................4! Results .............................................................................................................6! Ribosomal RNA content and Illumina library complexity ....................6! Assessment of sequencing platforms and assemblers for transcriptome assembly ........................................................................................7! Effect of sequencing depth on assembly coverage breadth and fragmentation ................................................................................8! Functional assessment of Geraniaceae nuclear transcriptomes .............9! Identification of selected organelle targeted genes ..............................10! Discussion .....................................................................................................11! Strategies for de novo assembly of transcriptomes ..............................11! Functional annotation of Geraniaceae transcriptomes .........................12! PPR proteins and sigma factors in Geraniaceae ..................................13! Conclusions ...................................................................................................14! Materials and Methods ..................................................................................15! RNA isolation ......................................................................................15! Illumina sequencing .............................................................................17! Roche/454 FLX sequencing .................................................................17! Read pre-processing .............................................................................18! Ribosomal RNA content and Illumina library complexity ..................18! Assembly .............................................................................................18! Comparative analysis of assemblies ....................................................19! Evaluation of assemblies with different proportion of reads ...............21! viii Orthologous genes identification .........................................................22! Functional annotation ...........................................................................22! Identification of selected organelle targeted genes ..............................22! Chapter 3: Coordinated rates of evolution between interacting plastid and nuclear genes in Geraniaceae .....................................................................................35! INTRODUCTION ........................................................................................35! RESULTS .....................................................................................................38! DISCUSSION ...............................................................................................43! Duplication and loss of sigma factor genes .........................................43! Coevolution of plastid and nuclear genomes .......................................44! METHODS ...................................................................................................48! RNA isolation, transcriptome sequencing and assembly .....................48! Identification of sigma factors .............................................................48! Phylogenetic analysis ...........................................................................48! Evolutionary rate estimation ................................................................49! Analysis of correlation of evolutionary rate ........................................49! Chapter 4: Coevolution between rates of nuclear encoded DNA RRR genes and plastid genome complexity ...........................................................................59!