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Title:Epigenetics and sex expression in trioecious papaya
Author(s):Aryal, Rishi
Director of Research:Ming, Ray R.
Doctoral Committee Chair(s):Ming, Ray R.
Doctoral Committee Member(s):Downie, Stephen R.; Vodkin, Lila O.; Hanzawa, Yoshie; Jacobs, Thomas W.
Department / Program:Plant Biology
Discipline:Plant Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):microRNA (miRNA)
small interfering RNA (siRNA)
small RNA
Purine-Pyramidine bias
Whole genome methylation
Papaya Ringspot Virus (PRSV)
Sex reversal
X-chromosome silencing
Abstract:Sex determination in papaya is regulated by a pair of recently evolved sex chromosomes. Genomic and genetic studies over the past century have made significant achievements towards the understanding of the sex determination system in papaya. However, the epigenetic aspects of sex determination and sex chromosome evolution in papaya have not yet been studied. To fill this gap, we analyzed the small non-coding RNAs (sRNA) and DNA methylation in different papaya sex types. Endogenous small RNA molecules of ~18-30 nucleotides play an important role in regulating gene expression and genome defense. Using large scale sequencing reads, we analyzed sRNA biogenesis and function in papaya. The sRNAs are processed from an RNA duplex by the RNase III enzyme dicer. Since the sRNA duplex is formed by Watson-Crick base pairing, equal proportion of purine-rich and pyrimidine-rich sequences are expected in the endogenous sRNA population. However, the endogenous sRNA population in papaya was biased towards the purine-rich sequences. Approximately 75% of the total sRNA population was represented by purine-rich sequences, while only ~25% of the sRNA sequences were pyrimidine-rich. Of the two purine bases, guanine was more prevalent in shorter sequences (22 and below) and adenine was more prevalent in the longer sequences (23 and above). Analysis of purine/pyrimidine distribution in the sRNA dataset from six other plant species (Arabidopsis thaliana, Populus trichocarpa, Medicago truncatula, Arachis hypogea, Glycine max, and Phaseolus vulgaris) provided similar results, indicating that the cell contains mechanisms to select the purine-rich strands from the sRNA duplex. The sRNA population in the cell was mostly represented by single copy sequences (~85% of the unique reads represented by a single copy sequences). Most of the single copy sequences were mapped to the overlapping regions of the genome, suggesting that they are processed from a single transcript by imprecise dicer processing. Only a small portion of the libraries was represented by multi-copy sequences. Using the sRNA reads mapped to the pericentromeric regions, we identified the location of the centromere on the papaya sex chromosomes. We observed the gradual increase in sRNA loci adjacent to the gaps on the sex chromosome physical maps, similar to the pattern observed in pericentromeric regions of other plant species. The sRNA molecules that aligned in the putative pericentromeric regions were highly conserved between the two chromosomes, providing the further evidence for the centromeric position on the sex chromosomes. The identified centromeric positions on the X and Y chromosomes are located about 1.6 mb apart relative to each other. Several intrachromosomal inversions on the papaya Y chromosome have been identified before. Our results show that the first inversion included the centromere on the Y chromosome, moving the centromere in reference to the centromere on the X chromosome. We identified 81 miRNAs in papaya using the sRNA deep sequencing reads. We observed the higher accumulation of antisense miRNA (miRNA*) strands of some miRNAs in virus infected leaves of papaya, suggesting that the miRNA* strands play functional role in stressed condition. Expression analysis identified the 12 miRNAs differentially expressed in the flowers of different sex types. The miRNAs that were expressed higher in male flowers were implicated in regulating the auxin signaling pathways. The miRNAs expressed higher in female flowers were those involved in regulating the embryo development and apical meristem identity. Using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), we analyzed the genome-wide DNA methylation in papaya. Higher methylation was observed in intergenic regions than in genic regions. We identified a total of 4,096 sex specific methylation sites on the papaya draft genome. Of these, 1,555 were specific to hermaphrodite, 1,399 were specific to male, and 1,145 were specific to female flowers. Comparing the methylation status of the papaya Y chromosomes showed the higher methylation of hermaphrodite specific region of Yh chromosome (HSY) than the male specific region of the Y chromosome (MSY). The HSY specific methylations were clustered in some locations of the chromosome compared to MSY specific methylation. The X chromosome showed higher methylation in female plants than in male and hermaphrodite plants, suggesting that the X chromosome is silenced in female plants. The distribution of female specific methylation sites on the X chromosome showed a similar pattern to the distribution of the X specific genes. We found five X specific genes methylated only in the female plants but the female specific methylation was not observed on the XY paired genes.
Issue Date:2013-05-24
Rights Information:Copyright 2013 Rishi Aryal
Date Available in IDEALS:2013-05-24
Date Deposited:2013-05

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