UC Riverside

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. 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 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 were 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.