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

ECHENIQUE VIVIANA; GARBUS INGRID; SELVA JUAN PABLO; ROMERO JOSE; MICHELETTO SANDRA; RODRIGO JUAN MANUEL; ZAPPACOSTA DIEGO; PELLINO MARCO; SHARBEL TIMOTHY

Rosario

Workshop; V Ciclo de Seminarios dobre avances en la Caracterización Genética y Molecular de la APOMIXIS; 2018

Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed byparthenogenetic development into offspring that are genetic clones of the mother plant. The aim of this work was to identifytranscripts 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 sexualgenotypes of this grass was constructed using total RNA from flowers at different developmental stages from the tetraploid genotypesTanganyika 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 of49,568 contigs (~80,000,000 bp) and 133,782 singletons (~40,000,000 bp). This information and previous sequenced ESTs of thisgrass were used to design a 1-million spotted chip array (Agilent) using the software provided by the manufacturer. Hybridizationswere preformed using RNA samples from four diplosporous tetraploid genotypes (Tanganyika, Don Walter, Ermelo and Morpa) andfrom four sexual genotypes (OTA (4x) and the diploids (2x) Victoria and accessions PI299920, PI208214). Resulting raw data fromthe Agilent's Feature Extraction (AFE) software were pre-processed and analyzed with GeneSpring GX, a multi-omics data analysissoftware. Preprocessing consisted on a percentile shift of 75% normalization, using the mean values of reproductive mode, apomicticor 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 thesexual one belong to the histone superfamily, 40S ribosomal transcripts and rRNA methyltransferases. The up-regulated genes (foldchange >2 and p values >0.01) in the apomictic genotypes compared with the sexual ones were mainly hypothetical proteins, some ofthem with the conserved domain MIF4G or belonging to the E3 ubiquitin ligase complex. Several cyclin isoforms, transcriptionfactors, F-box with WD repeats and leucine-rich repeats, B-box zinc finger, LRR receptor-like serine/threonine-protein kinase andDna J domains associated with hsp70 heat-shock system were all up-regulated in the apomictic genotypes. The most important resultof this work is the finding of three transcripts that are significantly more differentially expressed between apomictic and sexualgenotypes. These transcripts correspond to an unknown sequence that is exclusively present in apomictic genotypes, a cyclin and asplicing factor. We are actually working in order to establish the relationship of the expression of these genes and apomixis inEragrostis curvula.