ABSTRACT OF THE DISSERTATION
A Molecular Genetic Approach to Evaluate a Novel Seedless Phenotype Found in Tango, a New Variety of Mandarin Developed From Gamma-Irradiated W. Murcott
Jennifer Robyn Crowley
Doctor of Philosophy, Graduate Program in Genetics, Genomics, Bioinformatics University of California, Riverside, December 2011
Dr. Mikeal Roose, Chairperson
Since the beginning of its first known cultivation as described in ancient European literature (circa 300 BCE) citrus fruit has been one of the most important consumer products in the world. Currently, there is a great deal of research in developing new citrus varieties and in improving existing citrus varieties. Creating and improving citrus varieties can be achieved in one of several different ways: cross-breeding, genetic transformation, artificial selection, and irradiation. Molecular alterations within plant tissue exposed to radiation, such as genetic mutations, can and do occur, making the method of plant irradiation a desirable choice when attempting to create unique citrus varieties.
The intent of this study is to evaluate and compare the molecular genetics of one particular variety of citrus, a seedless mandarin-producing Tango, with its predecessor variety, a seedy mandarin-producing W. Murcott. The Tango variety was developed and propagated from gamma-irradiated W. Murcott budwood. In the case of Tango, fruit produced by a mature tree retained the same characteristics of size, color, easiness to peel, and sweetness as that produced by W. Murcott; Tango fruit differed from W. Murcott fruit only in its seedlessness.
To determine the underlying molecular differences between W. Murcott and Tango, molecular regions prone to alteration due to radiation exposure were targeted for analysis. These included chromosomes, LTR (long-terminal repeat) retrotransposon regions, and methylated/unmethylated CpG regions. For chromosome analysis, stained images of chromosome spreads taken from W. Murcott and Tango cells frozen in their meiotic metaphase state were captured via a Leica SP2 Confocal microscope. Images were compared and contrasted between the two citrus varieties to determine any significant differences in chromosome alignment. CIRE1, a sequenced, intact LTR retrotransposon in the Citrus sinensis genome was targeted for quantitative analysis in the W. Murcott and Tango genomes. Primer sets were designed based on the CIRE1 sequence found within the NCBI database archives, targeting three regions of CIRE1: a long-terminal repeat, an integrase gene, and a reverse transcriptase gene. CpG region methylation patterns were compared and contrasted between W. Murcott and Tango DNA extractions using LUMA analysis followed by pyrosequencing. HpaII (methylation insensitive) and MseI (methylation sensitive) restriction enzymes were used to digest W. Murcott and Tango DNA subsequent to pyrosequencing; the digestions were compared with their respective sequenced untreated controls as well as to each other to determine any significant deviations in CpG island methylation levels. Methylation-sensitive Amplified Fragment Length Polymorphism (ms-AFLP) analysis, which targeted methylated cytosine sites in the Citrus genome, was also performed by Lisa Mu to determine whether or not polymorphic bands between W. Murcott and Tango could be visualized on a polyacrylamide gel.
The results from experimentation showed that W. Murcott and Tango are nearly genetically identical. Both W. Murcott and Tango were found to have 14 copies of the CIRE1 retrotransposon in their genomes, and there were no significant differences in the percentage of methylated cytosines within their genomes (ranging from 53.1% to 58.6%) nor were there deviations in methylated cytosine patterns. However, there are some notable molecular genetic differences between the two varieties; in particular, chromosome misalignment in the meiotic metaphase state was detected in several images of Tango chromosome spreads, while only normal chromosome alignment in the meiotic metaphase state was ever detected in the images taken of W. Murcott chromosome spreads. This suggests that chromosomal abnormalities resulting from gamma radiation exposure could be the main underlying genetic cause of seedless fruit produced by Tango citrus plants.