UNIVERSITY OF CALIFORNIA RIVERSIDE Genomic Differences Between W. Murcott Mandarin and Its Mutational Derivative Tango A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant Biology by Yi Zhu June 2018 Dissertation Committee: Dr. Mikeal Roose, Chairperson Dr. Thomas Girke Dr. Patricia Springer Copyright by Yi Zhu 2018 The Dissertation of Yi Zhu is approved: Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS I would like to acknowledge all people who supported and devoted their effort to my research and dissertation. First of all, I truly appreciate Dr. Mikeal Roose for his long-term support and patient guidance throughout this dissertation project. I give many thanks to Dr. Claire Federici for providing technical supports and experimental materials that are related to my work, and managing the laboratory efficiently. I also would like to express my gratitude to my other dissertation committee members, Dr. Thomas Girke and Dr. Patricia Springer, for contributing considerable effort and time on my work during the whole process. I would like to thank Dr. Chandrika Ramadugu and Dr. Manjunath Keremane for their support on the use and data interpretation of Applied Biosystem HRM qPCR and CFX96 Real-Time PCR system, and extend many thanks to Dr. Chandrika Ramadugu for her generous assistance with DNA sequence and clones needed in my project. I am very thankful to Dr. Manuel Talon from IVIA for providing mutation information on the sequences, from which I could have a good start of this project. I also would like to thank Dr. Tim Williams and Dr. Jennifer Crowley for their previous valuable studies on Tango mandarin, based on which my project could proceed. Last, but not the least, I am grateful for my family’s support, as well as encouragement from my friends, Yuanhang Huang and Lu Yu, in the most difficult period. I would have given up halfway without you. iv DEDICATION This work is dedicated to my parents for their unending love and support. v ABSTRACT OF THE DISSERTATION Genomic Differences Between W. Murcott Mandarin and Its Mutational Derivative Tango by Yi Zhu Doctor of Philosophy, Graduate Program in Plant Biology University of California, Riverside, June 2018 Dr. Mikeal Roose, Chairperson The history of recorded citrus can be traced back to prior to 800 B.C. and citrus species and related genera are considered to be from the tropical and subtropical region from south- east Asia to north-eastern India, and southern China, Indo-Chinese peninsula and Malay Archipelago, and later spread to the Middle East by Arab traders and then the rest of the world. As one of the valuable consumer products, citrus has attracted great attention from researchers. Citrus breeding can create and/or improve citrus varieties using a variety of approaches, including cross-breeding, genetic transformation, mutation breeding, etc. Mutation breeding accelerates the process of mutation and provides a great opportunity to develop new traits of citrus. The purpose of this study is to identify the genomic differences between a very low-seeded citrus cultivar, Tango mandarin, and its seedy predecessor cultivar, W. Murcott mandarin. vi Tango mandarin was developed by gamma-irradiation mutation breeding of W. Murcott budwood. Both varieties share the same characteristics of size, color, sweetness and easiness of peeling, and they differ mainly in Tango having much lower pollen viability and in fruit seed content. W. Murcott fruit will be seedy unless grown in an isolated condition without cross-pollination while Tango is very low-seeded in all conditions. The intents of this study are to determine whether Tango is genetically uniform or has different genetic compositions within different cell layers, and to better characterize the putative cause of seedlessness of Tango fruit. To determine the underlying genomic differences between W. Murcott and Tango, Illumina next-generation sequence (NGS) data of both W. Murcott and Tango leaf DNA was primarily used in analysis. Sequence data was aligned to the haploid Clementine reference genome and SNPs and Indels were predicted with the Pindel and Dindel programs. Allele- specific PCR, high-resolution melting real-time PCR and TA-cloning followed by sequencing were used for verification. For chromosome rearrangement prediction, Illumina NGS data was analyzed by novoBreak program. Copy number variation was determined by analyzing the depth of coverage of Illumina NGS reads and by loss of heterozygosity analysis with Axiom® Citrus 15AX SNP array data. PCR with primers flanking the putative breakpoints followed by Sanger sequencing was used to verify the chromosome rearrangements and determine the position of chromosome rearrangement breakpoints. For chimerism analysis of Tango, Tango pollen and fruit albedo were used to represent cell layer II and juice vesicles were used to represent cell layer I. Previously vii verified short deletions and chromosome rearrangements were tested with DNA from these tissues to determine their genetic composition. These experiments identified and/or confirmed three SNPs and seven Indels which were heterozygous in Tango while they were homozygous in W. Murcott. These SNPs and Indels were developed into molecular markers that differentiate Tango and W. Murcott. The copy number variation results showed no large insertions or deletions present while one heterozygous translocation of a 6 Mb segment on chromosome 2 and one heterozygous inversion of 4 Mb on chromosome 4 in Tango were verified, and their breakpoints were determined. A potential translocation between chromosome 3 and chromosome 1 was also found. These two chromosome rearrangements could be responsible for misalignment and abnormal segregation of chromosomes during meiosis as previously observed, and the resulting inviable gametes could reduce seed content of fruits. Tango pollen and fruit tissue analysis proved that two short deletions and all chromosome rearrangements were present in cell layer II but absent in cell layer I of Tango meristem, while the other five short deletions may be present in both cell layer I and cell layer II. This suggested that cell layer II, which gives rise to gametes, was affected by the initial gamma irradiation, and inviable female gametes give rise to seedless Tango fruit and inviable male gametes greatly reduce male fertility. viii Table of Contents Abstract…………………………………………………………………………………..vi Chapter 1 Introduction………………………………………………………………………..1 References………………………………………………………………………..35 Chapter 2 Abstract…………………………………………………………………………..44 Introduction………………………………………………………………………46 Materials and Methods…………………………………………………………...65 Results……………………………………………………………………………75 Discussion………………………………………………………………………..81 References………………………………………………………………………..89 Figures and Tables……………………………………………………….……….96 Chapter 3 Abstract…………………………………………………………………………131 Introduction……………………………………………………………………..133 ix Materials and Methods………………………………………………………….147 Results…………………………………………………………………………..154 Discussion………………………………………………………………………168 References………………………………………………………………………180 Figures and Tables……………………………………………………………....184 Chapter 4 Abstract…………………………………………………………………………212 Introduction……………………………………………………………………..214 Materials and Methods………………………………………………………….230 Results…………………………………………………………………………..238 Discussion………………………………………………………………………244 References………………………………………………………………………253 Figures and Tables……………………………………………………………....258 Conclusion……………………………………………………………………………..279 x List of Figures Figure 2.1 DC-1 Short Deletion Forward Primer Design………………………..……...100 Figure 2.2 DC-7 Short Deletion Forward Primer Design………………………..……...101 Figure 2.3 3-GP SNP Forward Primer Design…………………………….....................102 Figure 2.4 5-GP SNP Forward Primer Design…………………………….....................103 Figure 2.5 33-GP SNP Reverse Primer Design……………………………....................104 Figure 2.6 DC-1 Real-Time PCR Primer Design…………………………….................105 Figure 2.7 DC-7 Real-Time PCR Primer Design…………………………….................106 Figure 2.8 Del2 Real-Time PCR Primer Design……………………………..................107 Figure 2.9 Del3 Real-Time PCR Primer Design……………………………..................108 Figure 2.10 Del4-2 Real-Time PCR Primer Design…………………………….............109 Figure 2.11 Del3-3 Real-Time PCR Primer Design…………………………….............110 Figure 2.12 Del2-3 Real-Time PCR Primer Design…………………………….............111 Figure 2.13 Allele-Specific PCR for DC-1 Short Deletion…………………………..…112 Figure 2.14 Allele-Specific PCR for DC-7 Short Deletion…………………………..…113 Figure 2.15 Allele-Specific PCR for 3-GP SNP……………………………..................114 xi Figure 2.16 Allele-Specific PCR for 5-GP SNP……………………………..................115 Figure 2.17 Allele-Specific PCR for 33-GP SNP………………………….…...............116 Figure 2.18 Real-Time PCR of DC-1 Short Deletion………………………..…….........117 Figure 2.19 Real-Time PCR of DC-7 Short Deletion………………………..…….........118 Figure 2.20 Real-Time PCR of Del2 Short Deletion………………………..……..........119 Figure 2.21 Real-Time PCR of Del3 Short Deletion………………………..……..........120 Figure 2.22 Real-Time PCR of Del4-2 Short Deletion…………………….……….......121 Figure 2.23 Real-Time PCR of Del3-3 Short Deletion…………………….……….......122 Figure 2.24 Real-Time PCR of Del2-3 Short Deletion…………………….……….......123 Figure 2.25 Alignment of Partial Sequences Including DC-1 Indel from Sequencing Result of TA-Cloning…………………………….......……………………………...................124 Figure 2.26 Alignment of Partial