Array-based comparative study of transcripts expressed in flowers of apomictic and sexual genotypes of Eragrostis curvula

Abstract

Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed by parthenogenetic development into offspring that are genetic clones of the mother plant. The aim of this work was to identify transcripts that are differentially expressed between apomictic and sexual genotypes of Eragrostis curvula by using the one-color (Cy3), 60-mer oligonucleotides microarray platform of Agilent. Previously, a floral reference transcriptome for apomictic and sexual genotypes of this grass was constructed using total RNAfrom flowers at different developmental stages from the tetraploid genotypes Tanganyika USDA (apomictic) and OTA USDA (sexual). Two samples from each genotype were collected (two different plants, biological replicas constituted by a mix of different developmental stages) and sequenced by using the 454 GS FLX+ Roche method, according to the protocol provided by the manufacturer. Transcripts de novo assembly (Newbler software package) gave a total of 49,568 contigs (~80,000,000 bp) and 133,782 singletons (~40,000,000 bp). This information and previous sequenced ESTs of this grass were used to design a 1-million spotted chip array (Agilent) using the software provided by the manufacturer. Hybridizations were preformed using RNAsamples from four diplosporous tetraploid genotypes (Tanganyika, Don Walter, Ermelo and Morpa) and from four sexual genotypes (OTA (4x) and the diploids (2x) Victoria and accessions PI299920, PI208214). Resulting raw data from the Agilent's Feature Extraction (AFE) software were pre-processed and analyzed with GeneSpring GX, a multi-omics data analysis software. Preprocessing consisted on a percentile shift of 75% normalization, using the mean values of reproductive mode, apomictic or sexual, as parameters. Subsequent analysis of the normalized data identified 110 transcripts differentially expressed (p-values <0.01) between the apomictic and sexual genotypes. Some of the genes that were repressed in the apomictic group compared with the sexual one belong to the histone superfamily, 40S ribosomal transcripts and rRNAmethyltransferases. The up-regulated genes (fold change >2 and p values >0.01) in the apomictic genotypes compared with the sexual ones were mainly hypothetical proteins, some of them with the conserved domain MIF4G or belonging to the E3 ubiquitin ligase complex. Several cyclin isoforms, transcription factors, F-box with WD repeats and leucine-rich repeats, B-box zinc finger, LRR receptor-like serine/threonine-protein kinase and Dna J domains associated with hsp70 heat-shock system were all up-regulated in the apomictic genotypes. The most important result of this work is the finding of three transcripts that are significantly more differentially expressed between apomictic and sexual genotypes. These transcripts correspond to an unknown sequence that is exclusively present in apomictic genotypes, a cyclin and a splicing factor. We are actually working in order to establish the relationship of the expression of these genes and apomixis in Eragrostis curvula.