LOCALIZATION OF THE RF3 RESTORER-OF-FERTILITY GENE FOR MAIZE S-TYPE CYTOPLASMIC MALE STERILITY A Dissertation Presented to The Faculty of the Graduate School At the University of Missouri In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy By TIFFANY LANGEWISCH Dr. Kathleen Newton, Dissertation Supervisor December 2012 © Copyright by Tiffany Langewisch 2012 All Rights Reserved The undersigned, appointed by the dean of the Graduate School, have examined the Dissertation entitled LOCATION OF THE RF3 RESTORER-OF-FERTILITY GENE FOR MAIZE S-TYPE CYTOPLASMIC MALE STERILTY Presented by TIFFANY LANGEWISCH A candidate for the degree of Doctor of Philosophy And hereby certify that, in their opinion, it is worthy of acceptance. Kathleen Newton James Birchler Sherry Flint-Garcia Chris Pires ACKNOWLEDGEMENTS I would first like to acknowledge members of my committee, past and present, including Drs. Kathleen Newton, James Birchler, Sherry Flint-Garcia, Michael McMullen, Christopher Pires, and Karen Cone. My committee has provided helpful input and guidance throughout my dissertation research. I would like to thank them for their invaluable advice in all aspects of my graduate studies. As my advisor, Kathleen Newton has particularly provided advice, guidance, and support. This research would not have been possible without the development of Rf3 near-isogenic lines by Dr. Susan Gabay-Laughnan. She has been a crucial resource for seed and feedback throughout the years. Barbara Sonderman is thanked for her tireless effort of keeping my “babies” happy and healthy in the greenhouse. I thankfully acknowledge everyone in the Newton lab for discussions, field assistance, and help in my everyday research. Specifically I would like to thank Kaitlyn Faries for her friendship, support, and various contributions to this project. Kate Guill is thanked for the preparation of material for whole-genome SNP genotyping. I thank Drs. Robert Gaeta and Kristen Leach for technical assistance in RNA work and quantitative real-time PCR (qRT-PCR) as well as Karen Bromert and the University of Missouri DNA Core for RNA-Seq library preparation and sequencing. Drs. William Spollen and Christopher Bottoms of the University of Missouri Bioinformatics Consortium in addition to Chad ii Neiderhuth and Saad Kahn were instrumental in my learning RNA-Seq. I also thank Zheng Wang, Kate Hertweck, and Dr. Sota Fujii for bioinformatics support. On a personal side, I would like to thank my parents for their love, support, and drive for me to continue my higher education. They have been a constant presence in both my personal life and my academic life, and I would not be here without them. Last, but certainly not least, I have a special thanks to my best friend and husband, Johnathan Shoff, who has experienced all the trials and tribulations of graduate school as an observer and constant listener. He has put up will all my “science” talk and has always been there with a smile to get me through the tough days. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................. ii LIST OF FIGURES ........................................................................................... x LIST OF TABLES ............................................................................................. xii LIST OF ABBREVIATIONS .............................................................................. xiii ABSTRACT ...................................................................................................... xvi Chapter 1. LITERATURE REVIEW ......................................................................... 1 1.1 Cytoplasmic Male Sterility ................................................................ 1 1.1.1 Importance of CMS in Agriculture .......................................... 2 1.1.2 Types of CMS in Maize .......................................................... 3 1.1.2.1 CMS Restoration ....................................................... 3 1.1.2.2 CMS-T ....................................................................... 3 1.1.2.3 CMS-C ....................................................................... 5 1.1.2.4 CMS-S ....................................................................... 7 1.1.3 Cause of CMS-S .................................................................... 8 1.2 Pentatricopeptide Repeat (PPR) Proteins ........................................ 9 1.2.1 Characteristics of PPR Proteins ............................................. 9 1.2.2 Classes of PPR Proteins ........................................................ 10 1.2.3 Roles of PPR Proteins in Plants ............................................. 11 iv 1.2.4 Expansion and Distribution of PPR Proteins .......................... 12 1.2.4.1 Expansion of PPR Proteins ........................................ 12 1.2.4.2 Clustering of PPR Proteins ........................................ 14 1.3 Restorer-of-Fertility Genes ............................................................... 15 1.3.1 Identification of Petunia Rf Gene ............................................ 15 1.3.2 Identification of Rice Rf1 Gene ............................................... 16 1.3.3 Exceptions to PPR Restorer Genes ....................................... 18 1.3.4 CMS-S Restorer Gene Rf3 ..................................................... 20 1.3.4.1 Location of Rf3 Locus on the Long Arm of Chromosome 2 .......................................................... 20 1.3.4.2 Mechanism of Restoration for Rf3 ............................. 21 2. FINE-MAPPING RF3 ............................................................................. 27 2.1 Introduction ...................................................................................... 27 2.2 Materials and Methods ..................................................................... 29 2.2.1 Creation of Rf3 Near-Isogenic Lines (NILs) ............................ 29 2.2.2 DNA Preparation .................................................................... 30 2.2.3 SCARE12M7 and CAPSE3P1 Markers .................................. 31 2.2.4 Simple Sequence Repeats (SSRs) Markers .......................... 33 2.2.5 Nested Association Mapping (NAM)....................................... 34 2.2.6 HapMap Single Nucleotide Polymorphism (SNP) Mapping .... 35 2.2.7 Infinium SNP Genotyping ....................................................... 37 v 2.3 Results ............................................................................................. 39 2.3.1 Sequencing SCARE12M7 and CAPSE3P1 Markers from Rf3 NILs ......................................................................... 39 2.3.2 Mapping Rf3 with SSRs ......................................................... 40 2.3.3 Mapping Rf3 with NAM ........................................................... 41 2.3.4 Mapping Rf3 with HapMap SNPs ........................................... 44 2.3.5 Mapping Rf3 with SNP Genotyping ........................................ 44 2.4 Discussion ....................................................................................... 45 3. CANDIDATE GENES FOR RF3 ............................................................ 65 3.1 Introduction ...................................................................................... 65 3.2 Material and Methods ...................................................................... 68 3.2.1 PPR Protein Bioinformatics .................................................... 68 3.2.1.1 Maize Homology to Rice Rf1a and Rf1b .................... 68 3.2.1.2 Identification of PPR Proteins on Contigs 108 and 109 ...................................................................... 69 3.2.1.3 Determination of Mitochondrial Targeting .................. 70 3.2.1.4 Classification of PPR Subclasses .............................. 71 3.2.1.5 Identification of Restorer-Like Proteins ...................... 72 3.2.2 Annotation of Rf3 Mapping Region ........................................ 72 3.2.3 Candidate Gene Approach ..................................................... 73 3.2.3.1 PCR Amplification ...................................................... 73 3.2.3.2 PCR Purification ........................................................ 75 3.2.3.3 Sequencing and Alignments ...................................... 76 vi 3.2.3.4 Location of PPR Candidates by Fluorescence in situ Hybridization (FISH) ........................................ 77 3.3 Results ............................................................................................. 80 3.3.1 Analysis of PPR Sequences on the Long Arm of Chromosome 2 ...................................................................... 80 3.3.2 Gene Content of Rf3 Region .................................................. 84 3.3.3 Analysis of PPR Candidates .................................................. 85 3.3.3.1 Criteria for PPR Candidates ....................................... 85 3.3.3.2 GRMZM2G450166 (PPR Candidate #1) .................... 86 3.3.3.3 GRMZM2G439814 (PPR Candidate #2) .................... 88 3.3.3.4 GRMZM2G453956 (PPR Candidate #3) .................... 89 3.3.3.5 GRMZM2G104286 (PPR Candidate #4) .................... 89 3.3.3.6 GRMZM2G416498 (PPR Candidate #5) .................... 91 3.3.3.7 GRMZM2G124602 (PPR Candidate #6) .................... 93 3.4 Discussion ....................................................................................... 94 4. RNA SEQUENCING .............................................................................